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M1 The three RNA polymerases characterizationM1 The three RNA polymerases characterization
and functionand function M2 RNA Pol genes the ribisomal repeatⅠM2 RNA Pol genes the ribisomal repeatⅠ M3 RNA Pol genes 5S and tRNA transcriptionⅢM3 RNA Pol genes 5S and tRNA transcriptionⅢ M4 RNA Pol genes promoters and enhancersⅡM4 RNA Pol genes promoters and enhancersⅡ M5 General transcription factors and RNA Pol ⅡM5 General transcription factors and RNA Pol Ⅱ initiationinitiation
Section MmdashTranscription in eukaryotes
Transcription and its control are much more complex in eukaryotes
There are 3 RNA polymerases each specific for a subset of RNAs
M1 The three RNA polymerases characterization and function
Eukaryotic RNA polymerasesThe mechanism of eukaryotic transcription is similar to that in prokaryotes However the large number of polypeptides associated with the eukaryotic transcription machinery makes it far more complex Three different RNA polymerase complexes are responsible for the transcription of different types of eukaryotic genes The different RNA polymerases were identified by chromatographic purification of the enzymes and elution at different salt concentrations(Topic B4) Each RNA polymerase has a different sensitivity to the fungal toxin α-amanitin and this can be used to distinguish their activities
RNA polynerase (RNA pol ) transcribes most rRNA genes It is located in the nⅠ Ⅰucleoli and is insensitive to α-amanitin
RNA polynerase (RNA pol ) transcribes all protein-coding genes and some smalⅡ Ⅱl nuclear RNA(snRNA) genes It is located in the nucleoplasm and is very sensitive to α-amanitin
RNA polynerase (RNA pol ) transcribes the genes for tRNA 5S rRNA U6 snRⅢ ⅢNA and certain other small RNAs It is located in the nucleoplasm and is moderately sensitive to α-amanitin
In addition to these nuclear enzymes eukaryotic cells contain additional polymerase in mitochondria and chloroplasts
mRNA
rRNA
tRNA
snRNA
scRNA
7S RNA
micro RNA
There Are Many Functional Classes of RNA
ndash 500-700kDa 12+ subunits most of RNA pol II structures are determined The genes encoding the two largest subunits of each RNA polymerase have homology to each other 1048766bull The largest subunits of each eukaryotic RNA polymerase is similar to the βrsquo subunit of the E coli polymerase and the second largest subunit is similar to the βsubunit which contains the actiove site of the E coli enzyme
bull Two subunits which are common to RNA Pol I and RNA PolIII and a further subunit which is specific to RNA Pol II have homology to the E coli RNA polymerase αsubunit
bull At least five other smaller subunits are common to the three different polymerases Each polymerase also contains an additional four to seven subunits which are only present in one type
RNA polymerase subunits
Similar Structures of Bacterial (left) and Eukaryotic (right) RNA Polymerases
Like bacterial RNA polymerases each of the eukaryotic enzymes catalyzes transcription in a 5rsquo to 3rsquo direction and synthesizes RNA complementary to the antisense template strand
bullThe reaction requires the precursor nuckeotides ATP GTPCTP and UTP and does not requires a primer for transcription initiation
bullThe purified eukaryotic RNA polymerases unlike the purified bacterial enzymes require the presence of additional initiation proteins before they are able to bind to promoters and initiate transcription
Eukaryotic RNA polymerase activities
The CTD of RNA Pol II
CTD---C-terminal domain RNA Pol II RPB1 subunit has (CTD) with repeat (YSPTSPS)n n=26-52
In vitro studies have shown that the CTD sequence may be phosphorylated at the serines and tyrosines
Phosphorylate Unphosphorylated
Unphosphorylated to initiate transcription Phosphorylated for elongation
M2 RNA Pol genes the ribosomal repeatⅠRNA polymerase ( RNA pol ) is responsible for the contiⅠ Ⅰnuous synthesis of rRNA during interphase Human cells contain five clusters of around 40 copie of rRNA gene situated on different chromosomes (see Fig1 and Topic D4)
Each rRNA gene produces a 45S rRNA transcript which is about 13000 nt long(see the Topic D4)
This transcript is cleaved to give one copy each of the 28S RNA (5000 nt) 18S(2000nt) and 58S (160 nt) rRNA (see Topic O1)
The continuous transcription of multiple gene copies of the RNAs is essential for sufficient production of the processed rRNAs which are packaged into ribosomes
Promoter
Transcription
Cleavage(the light pink regionsare degraded)
45S transcript
18S 58S 28S
5 318S 58S 28S
18S 58S 28SrRNA rRNA rRNA
Ribosomal RNA transcription units
Each rRNA cluster is known as a nucleolar organizer region since the nucleolus contains large loops of DNA correspondind to the gene clusters After a cell emerges from mitosis rRNA synthesis restarts and tiny nucleoli appear at the chromosomal locations of the rRNA genes
During active rRNA synthesis the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in the electron microscope as lsquoChrismas tree structuresrsquo
In these structures the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA
Short transcripts can be seen at the start of the gene which get longer until the end of the transcription unit which is indicated by disappearance of the RNA transcripts
Role of the nucleolus
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig2) The core element includes the transcription start site and encompasses bases -31 to +6 This sequence is essential for transcription
An additional element of around 50-80 bp named the upstream control element(UCE) begins about 100 bp upstream from the start site (-100)
The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
Transcription and its control are much more complex in eukaryotes
There are 3 RNA polymerases each specific for a subset of RNAs
M1 The three RNA polymerases characterization and function
Eukaryotic RNA polymerasesThe mechanism of eukaryotic transcription is similar to that in prokaryotes However the large number of polypeptides associated with the eukaryotic transcription machinery makes it far more complex Three different RNA polymerase complexes are responsible for the transcription of different types of eukaryotic genes The different RNA polymerases were identified by chromatographic purification of the enzymes and elution at different salt concentrations(Topic B4) Each RNA polymerase has a different sensitivity to the fungal toxin α-amanitin and this can be used to distinguish their activities
RNA polynerase (RNA pol ) transcribes most rRNA genes It is located in the nⅠ Ⅰucleoli and is insensitive to α-amanitin
RNA polynerase (RNA pol ) transcribes all protein-coding genes and some smalⅡ Ⅱl nuclear RNA(snRNA) genes It is located in the nucleoplasm and is very sensitive to α-amanitin
RNA polynerase (RNA pol ) transcribes the genes for tRNA 5S rRNA U6 snRⅢ ⅢNA and certain other small RNAs It is located in the nucleoplasm and is moderately sensitive to α-amanitin
In addition to these nuclear enzymes eukaryotic cells contain additional polymerase in mitochondria and chloroplasts
mRNA
rRNA
tRNA
snRNA
scRNA
7S RNA
micro RNA
There Are Many Functional Classes of RNA
ndash 500-700kDa 12+ subunits most of RNA pol II structures are determined The genes encoding the two largest subunits of each RNA polymerase have homology to each other 1048766bull The largest subunits of each eukaryotic RNA polymerase is similar to the βrsquo subunit of the E coli polymerase and the second largest subunit is similar to the βsubunit which contains the actiove site of the E coli enzyme
bull Two subunits which are common to RNA Pol I and RNA PolIII and a further subunit which is specific to RNA Pol II have homology to the E coli RNA polymerase αsubunit
bull At least five other smaller subunits are common to the three different polymerases Each polymerase also contains an additional four to seven subunits which are only present in one type
RNA polymerase subunits
Similar Structures of Bacterial (left) and Eukaryotic (right) RNA Polymerases
Like bacterial RNA polymerases each of the eukaryotic enzymes catalyzes transcription in a 5rsquo to 3rsquo direction and synthesizes RNA complementary to the antisense template strand
bullThe reaction requires the precursor nuckeotides ATP GTPCTP and UTP and does not requires a primer for transcription initiation
bullThe purified eukaryotic RNA polymerases unlike the purified bacterial enzymes require the presence of additional initiation proteins before they are able to bind to promoters and initiate transcription
Eukaryotic RNA polymerase activities
The CTD of RNA Pol II
CTD---C-terminal domain RNA Pol II RPB1 subunit has (CTD) with repeat (YSPTSPS)n n=26-52
In vitro studies have shown that the CTD sequence may be phosphorylated at the serines and tyrosines
Phosphorylate Unphosphorylated
Unphosphorylated to initiate transcription Phosphorylated for elongation
M2 RNA Pol genes the ribosomal repeatⅠRNA polymerase ( RNA pol ) is responsible for the contiⅠ Ⅰnuous synthesis of rRNA during interphase Human cells contain five clusters of around 40 copie of rRNA gene situated on different chromosomes (see Fig1 and Topic D4)
Each rRNA gene produces a 45S rRNA transcript which is about 13000 nt long(see the Topic D4)
This transcript is cleaved to give one copy each of the 28S RNA (5000 nt) 18S(2000nt) and 58S (160 nt) rRNA (see Topic O1)
The continuous transcription of multiple gene copies of the RNAs is essential for sufficient production of the processed rRNAs which are packaged into ribosomes
Promoter
Transcription
Cleavage(the light pink regionsare degraded)
45S transcript
18S 58S 28S
5 318S 58S 28S
18S 58S 28SrRNA rRNA rRNA
Ribosomal RNA transcription units
Each rRNA cluster is known as a nucleolar organizer region since the nucleolus contains large loops of DNA correspondind to the gene clusters After a cell emerges from mitosis rRNA synthesis restarts and tiny nucleoli appear at the chromosomal locations of the rRNA genes
During active rRNA synthesis the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in the electron microscope as lsquoChrismas tree structuresrsquo
In these structures the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA
Short transcripts can be seen at the start of the gene which get longer until the end of the transcription unit which is indicated by disappearance of the RNA transcripts
Role of the nucleolus
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig2) The core element includes the transcription start site and encompasses bases -31 to +6 This sequence is essential for transcription
An additional element of around 50-80 bp named the upstream control element(UCE) begins about 100 bp upstream from the start site (-100)
The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
Eukaryotic RNA polymerasesThe mechanism of eukaryotic transcription is similar to that in prokaryotes However the large number of polypeptides associated with the eukaryotic transcription machinery makes it far more complex Three different RNA polymerase complexes are responsible for the transcription of different types of eukaryotic genes The different RNA polymerases were identified by chromatographic purification of the enzymes and elution at different salt concentrations(Topic B4) Each RNA polymerase has a different sensitivity to the fungal toxin α-amanitin and this can be used to distinguish their activities
RNA polynerase (RNA pol ) transcribes most rRNA genes It is located in the nⅠ Ⅰucleoli and is insensitive to α-amanitin
RNA polynerase (RNA pol ) transcribes all protein-coding genes and some smalⅡ Ⅱl nuclear RNA(snRNA) genes It is located in the nucleoplasm and is very sensitive to α-amanitin
RNA polynerase (RNA pol ) transcribes the genes for tRNA 5S rRNA U6 snRⅢ ⅢNA and certain other small RNAs It is located in the nucleoplasm and is moderately sensitive to α-amanitin
In addition to these nuclear enzymes eukaryotic cells contain additional polymerase in mitochondria and chloroplasts
mRNA
rRNA
tRNA
snRNA
scRNA
7S RNA
micro RNA
There Are Many Functional Classes of RNA
ndash 500-700kDa 12+ subunits most of RNA pol II structures are determined The genes encoding the two largest subunits of each RNA polymerase have homology to each other 1048766bull The largest subunits of each eukaryotic RNA polymerase is similar to the βrsquo subunit of the E coli polymerase and the second largest subunit is similar to the βsubunit which contains the actiove site of the E coli enzyme
bull Two subunits which are common to RNA Pol I and RNA PolIII and a further subunit which is specific to RNA Pol II have homology to the E coli RNA polymerase αsubunit
bull At least five other smaller subunits are common to the three different polymerases Each polymerase also contains an additional four to seven subunits which are only present in one type
RNA polymerase subunits
Similar Structures of Bacterial (left) and Eukaryotic (right) RNA Polymerases
Like bacterial RNA polymerases each of the eukaryotic enzymes catalyzes transcription in a 5rsquo to 3rsquo direction and synthesizes RNA complementary to the antisense template strand
bullThe reaction requires the precursor nuckeotides ATP GTPCTP and UTP and does not requires a primer for transcription initiation
bullThe purified eukaryotic RNA polymerases unlike the purified bacterial enzymes require the presence of additional initiation proteins before they are able to bind to promoters and initiate transcription
Eukaryotic RNA polymerase activities
The CTD of RNA Pol II
CTD---C-terminal domain RNA Pol II RPB1 subunit has (CTD) with repeat (YSPTSPS)n n=26-52
In vitro studies have shown that the CTD sequence may be phosphorylated at the serines and tyrosines
Phosphorylate Unphosphorylated
Unphosphorylated to initiate transcription Phosphorylated for elongation
M2 RNA Pol genes the ribosomal repeatⅠRNA polymerase ( RNA pol ) is responsible for the contiⅠ Ⅰnuous synthesis of rRNA during interphase Human cells contain five clusters of around 40 copie of rRNA gene situated on different chromosomes (see Fig1 and Topic D4)
Each rRNA gene produces a 45S rRNA transcript which is about 13000 nt long(see the Topic D4)
This transcript is cleaved to give one copy each of the 28S RNA (5000 nt) 18S(2000nt) and 58S (160 nt) rRNA (see Topic O1)
The continuous transcription of multiple gene copies of the RNAs is essential for sufficient production of the processed rRNAs which are packaged into ribosomes
Promoter
Transcription
Cleavage(the light pink regionsare degraded)
45S transcript
18S 58S 28S
5 318S 58S 28S
18S 58S 28SrRNA rRNA rRNA
Ribosomal RNA transcription units
Each rRNA cluster is known as a nucleolar organizer region since the nucleolus contains large loops of DNA correspondind to the gene clusters After a cell emerges from mitosis rRNA synthesis restarts and tiny nucleoli appear at the chromosomal locations of the rRNA genes
During active rRNA synthesis the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in the electron microscope as lsquoChrismas tree structuresrsquo
In these structures the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA
Short transcripts can be seen at the start of the gene which get longer until the end of the transcription unit which is indicated by disappearance of the RNA transcripts
Role of the nucleolus
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig2) The core element includes the transcription start site and encompasses bases -31 to +6 This sequence is essential for transcription
An additional element of around 50-80 bp named the upstream control element(UCE) begins about 100 bp upstream from the start site (-100)
The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
mRNA
rRNA
tRNA
snRNA
scRNA
7S RNA
micro RNA
There Are Many Functional Classes of RNA
ndash 500-700kDa 12+ subunits most of RNA pol II structures are determined The genes encoding the two largest subunits of each RNA polymerase have homology to each other 1048766bull The largest subunits of each eukaryotic RNA polymerase is similar to the βrsquo subunit of the E coli polymerase and the second largest subunit is similar to the βsubunit which contains the actiove site of the E coli enzyme
bull Two subunits which are common to RNA Pol I and RNA PolIII and a further subunit which is specific to RNA Pol II have homology to the E coli RNA polymerase αsubunit
bull At least five other smaller subunits are common to the three different polymerases Each polymerase also contains an additional four to seven subunits which are only present in one type
RNA polymerase subunits
Similar Structures of Bacterial (left) and Eukaryotic (right) RNA Polymerases
Like bacterial RNA polymerases each of the eukaryotic enzymes catalyzes transcription in a 5rsquo to 3rsquo direction and synthesizes RNA complementary to the antisense template strand
bullThe reaction requires the precursor nuckeotides ATP GTPCTP and UTP and does not requires a primer for transcription initiation
bullThe purified eukaryotic RNA polymerases unlike the purified bacterial enzymes require the presence of additional initiation proteins before they are able to bind to promoters and initiate transcription
Eukaryotic RNA polymerase activities
The CTD of RNA Pol II
CTD---C-terminal domain RNA Pol II RPB1 subunit has (CTD) with repeat (YSPTSPS)n n=26-52
In vitro studies have shown that the CTD sequence may be phosphorylated at the serines and tyrosines
Phosphorylate Unphosphorylated
Unphosphorylated to initiate transcription Phosphorylated for elongation
M2 RNA Pol genes the ribosomal repeatⅠRNA polymerase ( RNA pol ) is responsible for the contiⅠ Ⅰnuous synthesis of rRNA during interphase Human cells contain five clusters of around 40 copie of rRNA gene situated on different chromosomes (see Fig1 and Topic D4)
Each rRNA gene produces a 45S rRNA transcript which is about 13000 nt long(see the Topic D4)
This transcript is cleaved to give one copy each of the 28S RNA (5000 nt) 18S(2000nt) and 58S (160 nt) rRNA (see Topic O1)
The continuous transcription of multiple gene copies of the RNAs is essential for sufficient production of the processed rRNAs which are packaged into ribosomes
Promoter
Transcription
Cleavage(the light pink regionsare degraded)
45S transcript
18S 58S 28S
5 318S 58S 28S
18S 58S 28SrRNA rRNA rRNA
Ribosomal RNA transcription units
Each rRNA cluster is known as a nucleolar organizer region since the nucleolus contains large loops of DNA correspondind to the gene clusters After a cell emerges from mitosis rRNA synthesis restarts and tiny nucleoli appear at the chromosomal locations of the rRNA genes
During active rRNA synthesis the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in the electron microscope as lsquoChrismas tree structuresrsquo
In these structures the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA
Short transcripts can be seen at the start of the gene which get longer until the end of the transcription unit which is indicated by disappearance of the RNA transcripts
Role of the nucleolus
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig2) The core element includes the transcription start site and encompasses bases -31 to +6 This sequence is essential for transcription
An additional element of around 50-80 bp named the upstream control element(UCE) begins about 100 bp upstream from the start site (-100)
The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
ndash 500-700kDa 12+ subunits most of RNA pol II structures are determined The genes encoding the two largest subunits of each RNA polymerase have homology to each other 1048766bull The largest subunits of each eukaryotic RNA polymerase is similar to the βrsquo subunit of the E coli polymerase and the second largest subunit is similar to the βsubunit which contains the actiove site of the E coli enzyme
bull Two subunits which are common to RNA Pol I and RNA PolIII and a further subunit which is specific to RNA Pol II have homology to the E coli RNA polymerase αsubunit
bull At least five other smaller subunits are common to the three different polymerases Each polymerase also contains an additional four to seven subunits which are only present in one type
RNA polymerase subunits
Similar Structures of Bacterial (left) and Eukaryotic (right) RNA Polymerases
Like bacterial RNA polymerases each of the eukaryotic enzymes catalyzes transcription in a 5rsquo to 3rsquo direction and synthesizes RNA complementary to the antisense template strand
bullThe reaction requires the precursor nuckeotides ATP GTPCTP and UTP and does not requires a primer for transcription initiation
bullThe purified eukaryotic RNA polymerases unlike the purified bacterial enzymes require the presence of additional initiation proteins before they are able to bind to promoters and initiate transcription
Eukaryotic RNA polymerase activities
The CTD of RNA Pol II
CTD---C-terminal domain RNA Pol II RPB1 subunit has (CTD) with repeat (YSPTSPS)n n=26-52
In vitro studies have shown that the CTD sequence may be phosphorylated at the serines and tyrosines
Phosphorylate Unphosphorylated
Unphosphorylated to initiate transcription Phosphorylated for elongation
M2 RNA Pol genes the ribosomal repeatⅠRNA polymerase ( RNA pol ) is responsible for the contiⅠ Ⅰnuous synthesis of rRNA during interphase Human cells contain five clusters of around 40 copie of rRNA gene situated on different chromosomes (see Fig1 and Topic D4)
Each rRNA gene produces a 45S rRNA transcript which is about 13000 nt long(see the Topic D4)
This transcript is cleaved to give one copy each of the 28S RNA (5000 nt) 18S(2000nt) and 58S (160 nt) rRNA (see Topic O1)
The continuous transcription of multiple gene copies of the RNAs is essential for sufficient production of the processed rRNAs which are packaged into ribosomes
Promoter
Transcription
Cleavage(the light pink regionsare degraded)
45S transcript
18S 58S 28S
5 318S 58S 28S
18S 58S 28SrRNA rRNA rRNA
Ribosomal RNA transcription units
Each rRNA cluster is known as a nucleolar organizer region since the nucleolus contains large loops of DNA correspondind to the gene clusters After a cell emerges from mitosis rRNA synthesis restarts and tiny nucleoli appear at the chromosomal locations of the rRNA genes
During active rRNA synthesis the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in the electron microscope as lsquoChrismas tree structuresrsquo
In these structures the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA
Short transcripts can be seen at the start of the gene which get longer until the end of the transcription unit which is indicated by disappearance of the RNA transcripts
Role of the nucleolus
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig2) The core element includes the transcription start site and encompasses bases -31 to +6 This sequence is essential for transcription
An additional element of around 50-80 bp named the upstream control element(UCE) begins about 100 bp upstream from the start site (-100)
The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
Similar Structures of Bacterial (left) and Eukaryotic (right) RNA Polymerases
Like bacterial RNA polymerases each of the eukaryotic enzymes catalyzes transcription in a 5rsquo to 3rsquo direction and synthesizes RNA complementary to the antisense template strand
bullThe reaction requires the precursor nuckeotides ATP GTPCTP and UTP and does not requires a primer for transcription initiation
bullThe purified eukaryotic RNA polymerases unlike the purified bacterial enzymes require the presence of additional initiation proteins before they are able to bind to promoters and initiate transcription
Eukaryotic RNA polymerase activities
The CTD of RNA Pol II
CTD---C-terminal domain RNA Pol II RPB1 subunit has (CTD) with repeat (YSPTSPS)n n=26-52
In vitro studies have shown that the CTD sequence may be phosphorylated at the serines and tyrosines
Phosphorylate Unphosphorylated
Unphosphorylated to initiate transcription Phosphorylated for elongation
M2 RNA Pol genes the ribosomal repeatⅠRNA polymerase ( RNA pol ) is responsible for the contiⅠ Ⅰnuous synthesis of rRNA during interphase Human cells contain five clusters of around 40 copie of rRNA gene situated on different chromosomes (see Fig1 and Topic D4)
Each rRNA gene produces a 45S rRNA transcript which is about 13000 nt long(see the Topic D4)
This transcript is cleaved to give one copy each of the 28S RNA (5000 nt) 18S(2000nt) and 58S (160 nt) rRNA (see Topic O1)
The continuous transcription of multiple gene copies of the RNAs is essential for sufficient production of the processed rRNAs which are packaged into ribosomes
Promoter
Transcription
Cleavage(the light pink regionsare degraded)
45S transcript
18S 58S 28S
5 318S 58S 28S
18S 58S 28SrRNA rRNA rRNA
Ribosomal RNA transcription units
Each rRNA cluster is known as a nucleolar organizer region since the nucleolus contains large loops of DNA correspondind to the gene clusters After a cell emerges from mitosis rRNA synthesis restarts and tiny nucleoli appear at the chromosomal locations of the rRNA genes
During active rRNA synthesis the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in the electron microscope as lsquoChrismas tree structuresrsquo
In these structures the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA
Short transcripts can be seen at the start of the gene which get longer until the end of the transcription unit which is indicated by disappearance of the RNA transcripts
Role of the nucleolus
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig2) The core element includes the transcription start site and encompasses bases -31 to +6 This sequence is essential for transcription
An additional element of around 50-80 bp named the upstream control element(UCE) begins about 100 bp upstream from the start site (-100)
The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
Like bacterial RNA polymerases each of the eukaryotic enzymes catalyzes transcription in a 5rsquo to 3rsquo direction and synthesizes RNA complementary to the antisense template strand
bullThe reaction requires the precursor nuckeotides ATP GTPCTP and UTP and does not requires a primer for transcription initiation
bullThe purified eukaryotic RNA polymerases unlike the purified bacterial enzymes require the presence of additional initiation proteins before they are able to bind to promoters and initiate transcription
Eukaryotic RNA polymerase activities
The CTD of RNA Pol II
CTD---C-terminal domain RNA Pol II RPB1 subunit has (CTD) with repeat (YSPTSPS)n n=26-52
In vitro studies have shown that the CTD sequence may be phosphorylated at the serines and tyrosines
Phosphorylate Unphosphorylated
Unphosphorylated to initiate transcription Phosphorylated for elongation
M2 RNA Pol genes the ribosomal repeatⅠRNA polymerase ( RNA pol ) is responsible for the contiⅠ Ⅰnuous synthesis of rRNA during interphase Human cells contain five clusters of around 40 copie of rRNA gene situated on different chromosomes (see Fig1 and Topic D4)
Each rRNA gene produces a 45S rRNA transcript which is about 13000 nt long(see the Topic D4)
This transcript is cleaved to give one copy each of the 28S RNA (5000 nt) 18S(2000nt) and 58S (160 nt) rRNA (see Topic O1)
The continuous transcription of multiple gene copies of the RNAs is essential for sufficient production of the processed rRNAs which are packaged into ribosomes
Promoter
Transcription
Cleavage(the light pink regionsare degraded)
45S transcript
18S 58S 28S
5 318S 58S 28S
18S 58S 28SrRNA rRNA rRNA
Ribosomal RNA transcription units
Each rRNA cluster is known as a nucleolar organizer region since the nucleolus contains large loops of DNA correspondind to the gene clusters After a cell emerges from mitosis rRNA synthesis restarts and tiny nucleoli appear at the chromosomal locations of the rRNA genes
During active rRNA synthesis the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in the electron microscope as lsquoChrismas tree structuresrsquo
In these structures the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA
Short transcripts can be seen at the start of the gene which get longer until the end of the transcription unit which is indicated by disappearance of the RNA transcripts
Role of the nucleolus
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig2) The core element includes the transcription start site and encompasses bases -31 to +6 This sequence is essential for transcription
An additional element of around 50-80 bp named the upstream control element(UCE) begins about 100 bp upstream from the start site (-100)
The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
The CTD of RNA Pol II
CTD---C-terminal domain RNA Pol II RPB1 subunit has (CTD) with repeat (YSPTSPS)n n=26-52
In vitro studies have shown that the CTD sequence may be phosphorylated at the serines and tyrosines
Phosphorylate Unphosphorylated
Unphosphorylated to initiate transcription Phosphorylated for elongation
M2 RNA Pol genes the ribosomal repeatⅠRNA polymerase ( RNA pol ) is responsible for the contiⅠ Ⅰnuous synthesis of rRNA during interphase Human cells contain five clusters of around 40 copie of rRNA gene situated on different chromosomes (see Fig1 and Topic D4)
Each rRNA gene produces a 45S rRNA transcript which is about 13000 nt long(see the Topic D4)
This transcript is cleaved to give one copy each of the 28S RNA (5000 nt) 18S(2000nt) and 58S (160 nt) rRNA (see Topic O1)
The continuous transcription of multiple gene copies of the RNAs is essential for sufficient production of the processed rRNAs which are packaged into ribosomes
Promoter
Transcription
Cleavage(the light pink regionsare degraded)
45S transcript
18S 58S 28S
5 318S 58S 28S
18S 58S 28SrRNA rRNA rRNA
Ribosomal RNA transcription units
Each rRNA cluster is known as a nucleolar organizer region since the nucleolus contains large loops of DNA correspondind to the gene clusters After a cell emerges from mitosis rRNA synthesis restarts and tiny nucleoli appear at the chromosomal locations of the rRNA genes
During active rRNA synthesis the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in the electron microscope as lsquoChrismas tree structuresrsquo
In these structures the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA
Short transcripts can be seen at the start of the gene which get longer until the end of the transcription unit which is indicated by disappearance of the RNA transcripts
Role of the nucleolus
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig2) The core element includes the transcription start site and encompasses bases -31 to +6 This sequence is essential for transcription
An additional element of around 50-80 bp named the upstream control element(UCE) begins about 100 bp upstream from the start site (-100)
The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
M2 RNA Pol genes the ribosomal repeatⅠRNA polymerase ( RNA pol ) is responsible for the contiⅠ Ⅰnuous synthesis of rRNA during interphase Human cells contain five clusters of around 40 copie of rRNA gene situated on different chromosomes (see Fig1 and Topic D4)
Each rRNA gene produces a 45S rRNA transcript which is about 13000 nt long(see the Topic D4)
This transcript is cleaved to give one copy each of the 28S RNA (5000 nt) 18S(2000nt) and 58S (160 nt) rRNA (see Topic O1)
The continuous transcription of multiple gene copies of the RNAs is essential for sufficient production of the processed rRNAs which are packaged into ribosomes
Promoter
Transcription
Cleavage(the light pink regionsare degraded)
45S transcript
18S 58S 28S
5 318S 58S 28S
18S 58S 28SrRNA rRNA rRNA
Ribosomal RNA transcription units
Each rRNA cluster is known as a nucleolar organizer region since the nucleolus contains large loops of DNA correspondind to the gene clusters After a cell emerges from mitosis rRNA synthesis restarts and tiny nucleoli appear at the chromosomal locations of the rRNA genes
During active rRNA synthesis the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in the electron microscope as lsquoChrismas tree structuresrsquo
In these structures the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA
Short transcripts can be seen at the start of the gene which get longer until the end of the transcription unit which is indicated by disappearance of the RNA transcripts
Role of the nucleolus
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig2) The core element includes the transcription start site and encompasses bases -31 to +6 This sequence is essential for transcription
An additional element of around 50-80 bp named the upstream control element(UCE) begins about 100 bp upstream from the start site (-100)
The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
Promoter
Transcription
Cleavage(the light pink regionsare degraded)
45S transcript
18S 58S 28S
5 318S 58S 28S
18S 58S 28SrRNA rRNA rRNA
Ribosomal RNA transcription units
Each rRNA cluster is known as a nucleolar organizer region since the nucleolus contains large loops of DNA correspondind to the gene clusters After a cell emerges from mitosis rRNA synthesis restarts and tiny nucleoli appear at the chromosomal locations of the rRNA genes
During active rRNA synthesis the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in the electron microscope as lsquoChrismas tree structuresrsquo
In these structures the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA
Short transcripts can be seen at the start of the gene which get longer until the end of the transcription unit which is indicated by disappearance of the RNA transcripts
Role of the nucleolus
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig2) The core element includes the transcription start site and encompasses bases -31 to +6 This sequence is essential for transcription
An additional element of around 50-80 bp named the upstream control element(UCE) begins about 100 bp upstream from the start site (-100)
The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
Each rRNA cluster is known as a nucleolar organizer region since the nucleolus contains large loops of DNA correspondind to the gene clusters After a cell emerges from mitosis rRNA synthesis restarts and tiny nucleoli appear at the chromosomal locations of the rRNA genes
During active rRNA synthesis the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in the electron microscope as lsquoChrismas tree structuresrsquo
In these structures the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA
Short transcripts can be seen at the start of the gene which get longer until the end of the transcription unit which is indicated by disappearance of the RNA transcripts
Role of the nucleolus
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig2) The core element includes the transcription start site and encompasses bases -31 to +6 This sequence is essential for transcription
An additional element of around 50-80 bp named the upstream control element(UCE) begins about 100 bp upstream from the start site (-100)
The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig2) The core element includes the transcription start site and encompasses bases -31 to +6 This sequence is essential for transcription
An additional element of around 50-80 bp named the upstream control element(UCE) begins about 100 bp upstream from the start site (-100)
The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
UBF A specific DNA-binding proteincalled upstream binding factor binds to the UCESL1 An additional factor called selectivity factor 1 is essential for RNA Pol transcription SL1 binds tⅠo and stabilizes the UBF-DNA complex and interacts with free downstream part of the core elementTBP One of the subunits of SL1 called TATA-binding protein is required for initiation by all three eukaryotic RNA polymerases
TAFI s（ TBP-associated factors） as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs
Upstream binding factor
Schematic model for rRNA transcription initiation
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
M3 RNA Pol Genes 5S and tRNA traⅢnscription
RNA polymerase (RNA poly ) is a complex of at Ⅲ Ⅲ16 defferent subunits Like RNA Pol it is located iⅡn the nucleoplasm
RNA polymerase synthesizes the precursors of 5S ⅢrRNA the tRNAs and snRNA and cytosolic RNAs
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
tRNA gene transcription
Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences
A box 5rsquo-TGGCNNAGTGG-3rsquo B box 5rsquo-GGTTCGANNCC-3rsquo
TFIIIB and TFIIIC are required for tRNA gene transcription
bull TFIIIB allows RNA Poly to binⅢd and initiate transcription
bull TFIIIC is an assembly factor for the positioning of the initiation factor TF BⅢ
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
5S rRNA gene transcription
Initiation of transcription at a eukaryotic 5S rRNA promoter
The promoters of the 5S rRNA genes contain C box and A box as internal control regions
5S rRNA transcription initiation needs an additional assembly factor TF AⅢ relative to the tRNA transcription initiation TF A Ⅲ acts to bind to C box and stabilize the interaction between TF C Ⅲ and 5S rRNA
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites The coding region of the U6 snRNA has a characteristic A box However this sequence is not required for transcription The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters including a TATA box at bases -30 to -23 these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes
Alternative RNA Pol promoters and RⅢNA Pol terminationⅢ
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
RNA Pol terminationⅢ
Termination of transcription by RNA Pol appears oⅢnly to require polymerase recognition of a simple nucleotide sequence consisting of dA residues whose termination efficiency is affected by surrounding sequence
Thus the sequence 5rsquo-GCAAAAGC-3rsquo is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
M4 RNA Pol II genes promoters and enhancers
RNA Polymerase II (RNA Pol II) is located in the nucleoplasm It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes
The pre-mRNAs must be processed after synthesis by cap formation at the 5rsquo-end of the RNA and poly (A) addition at the 3rsquo-end as well as removal of introns by splicing
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
TATA box
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences TATAAAA
GC + CAAT boxesndash100
ndash50 ndash25 +1Py2CAPy5
Promoters
TATA box Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription It has the 7 bp consensus sequence 5rsquo-TATA（ AT） A（ AT） -3rsquo although it is now known that the protein which binds to the TATA box TBP binds to an 8 bp sequence that includes an additional downstream base pair whose identity is not important
Initiator element The initiator element is located around the transcription strt site Many initiator elements have a C at -1 and A at +1
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
Upstream regulatory elements
These elements are found in many genes which vary widely in their levels of expression in different tissues
Two common examples are SP1 box which is found upstream of many genes both with and without TATA boxes and the CCAAT box
Promoters may have one both or multiple copies of these sequences
These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site This kind of elements are called as enhancer Classically enhancers have the following general chracteristics
bull They exert strong activation of transcription of a linked gene from the correct start sitebullThey activate transcription when placed in either orientation with respect to linked genesbullThey are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start sitebullThey exert preferential stimulation of the closest of two tandem promoters
Enhancers
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
M5 General transcription factors and RNA Pol II initiation
RNA Pol II basal transcription fatorsA serial of nuclear transcription factors have been identified purified amd cloned These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA TFIIB TFIIC TFIID They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation
TFIID Inn promoters containing a TATA box the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex It seems that in mammalian cells TBP binds to the TATA box and is then joined by at least eight 由 TBP和 TAFIIs to form TFIID
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
TBP
TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation TBP is a monomeric protein with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
TBP structureTBP has been shown to have saddle structure with an overall dyad symmetry but two halves of the molecule are not identical TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound This results in a kink of about 45deg between the first two and last two base pariss of the 8 bp TATA element
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
TFIIA TFIIB and RNA polymearse binding
TFIIA TFIIA binds to TFIID and enhances TFIID binding to the TATA box stabilizing the TFIID-DNA complex TFIIA is made up of at least three subunits
TFIIB and RNA polymearse binding Once TFIID has bound to the DNA another transcription factor TFIIB binds to TFIID TFIIB can also bind to the RNA polymerase This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator TFIIF
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
Factors binding after RNA polymerase
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
These proteins are necessary for transcription in vitro and associate with the complex in a defined order
TFIIH is a large compex which is made up of at least five subunits TFIIJ remains to fully characterized
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase
This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
TFIIH therefore seems to have a very important function in control of transcriptiom elongation
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIIB binds to TFIID
TFIIB acts as a bridge to bind toRNA polymerase IITFIIF
TFIIE and TFIIH bindto RNA polymerase II
RNA polymerase
TFIIB
TFIIA
TFIIA
TFIIA binds to TFIID and enhances TFIID binding to TATA box
Once TFIID has bound to the DNA TFIIB binds to TFIID
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
After RNA polymerase binding three other transcription factors TFIIE TFIIH and TFIIJ rapidly asociate with the compex
Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region
Transcription factors
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
The initiator transcription complex
Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site
It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element
TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
Vmax = 50 nts sec
1048766 About 110th DNA polymerase1048766 Which needs to be fast1048766 Initiated at very few points1048766 Only ~ 10 DNA pol molecules cell
1048766 Each has to be very fast1048766 ~3000 molecules cell1048766 Transcription simultaneously at many points
RNA pol Speed
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
Error rate of ~ 10-4 to 10-51048766 Much greater than DNA pol1048766 Less than expected just from W-C base-pairing1048766 Suggests proof-reading Details of proof-reading not understood
RNA pol Fidelity
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
What about the dsDNA sequence tells RNA polymerase where to start
Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer
Homework
![[V]. Process of Transcription and Transcriptional Control of Gene Expression 1 RNA polymerases and Initiation of transcription Transcriptional elongation](https://img.pdfslide.us/doc/110x75/56649e595503460f94b52b31/v-process-of-transcription-and-transcriptional-control-of-gene-expression.jpg)
