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Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
Section M Transcription in Eukaryotes
M1 The three RNA polmerases: Characterization and functionM2 RNA pol. I genes: The ribosomal repeatM3 RNA pol. III genes: tRNA and 5S ribosomal repeatM4 RNA pol. II genes: Promoters and enhancersM5 General transcri. factors and pol. II initiation
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
M1 The three RNA polmerases:Characterization and function
• Eukaryotic RNA polymerases
• Eukaryotic RNA polymerases activities
• RNA polymerase subunit
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
Eukaryotic RNA polymerases
Classification: Three eukaryotic polymerases transcribe different sets of genes. Their activities are distinguished by their different sensitivities to the fungal toxin -amanitin:
• RNA pol I: transcribes most rRNA genes. It is located in the nucleoli and is insensitive to -amanitin.
• RNA pol II: transcribes all protein-coding genes and some small nuclear RNA (snRNA) genes. It is located in the nucleoplasm and is very sensitive to -amanitin.
• RNA pol III: transcribes the genes for tRNA, 5S rRNA, U6 snRNA and certain other small RNAs. It is located in the nucleoplasm and is moderately sensitive to -amanitin.
In addition to these nuclear enzymes, eukaryotic cells contain
additional RNA pol in mitochondria and chloroplasts.
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
Eukaryotic RNA polymerases
Transcription
for
Location
in
Sensitivity to
-amanitin
RNA pol Imost of rRNA genes
nucleoli insensitive
( - )
RNA pol IImRNA and some snRNA
nucleoplasm very sensitive (+ +)
RNA pol IIItRNA and
5S rRNA
nucleoplasm moderately sensitive (+)
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
Activities of three RNA pol.Transcription in Eukaryotes
Transcripition
of rRNA gene
Transcripition of tRNA and 5S rRNA gene
Transcripition of mRNA and snRNA genes
RNA Pol I RNA Pol II RNA Pol III
mRNA
RNA polymerase subunit
RNA pol. I, II, III
L’ with 78% ± homologous
among 3 RNA pol. (I, II, III)
L ~ β of prokaryote RNA pol
L’~ β’ of prokaryote RNA pol;
Including L, L’ subunit & 7-12 small subunits
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
M2 RNA pol. I genes: The ribosomal repeat
• Ribosomal RNA genes• Promoter of RNA pol. I• Upstream binding factor• Selectivity factor 1• TBP and TAFIs• Other rRNA genes
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
rRNA genes• RNA pol I is responsible for the continuous synthesis of
rRNAs during inter-phase. • Human cells contain 5 clusters of around 40 copies of the
rRNA gene situated in different chromosomes.• Each rRNA gene produces a 45S rRNA transcript which is
about 13 000 nt long.
18S 28S
5.8S
Singletranscr. U
Tandem array
5‘ 3‘
Untranscribed spacer DNA
• This transcript is cleaved to give each copy of the 28S RNA (5000 nt), 18S (2000 nt) and 5.8S (160 nt) rRNAs.
Section M: Transcription in Eukaryotes Yang Xu, College of Life SciencesSection O: RNA Processing and RNPs. 华中师范大学生科院 杨旭
Eukaryotic ribosomesThe complete mammalian (rat) 80S ribosome is composed of one
large 60S subunit and one small 40S subunit. The 40S subunit contains an 18S rRNA molecule and about 30 distinct proteins. The 60S subunit contains one 5S rRNA, one 5.8S rRNA, one 28S rRNA and about 45 proteins
5 S(120 bases)
+
ProteinsL1, L2, L3….L45
Sub-units
60 S
18S(1900 bases)
+
S1, S2, S3….S30
r RNA
40S
28S 5.8S(4800+160bases)
80 S
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
Promoter of RNA pol. I Pre-rRNA gene promoters: In mammalian, pre-rRNA gene
promoters have a bipartite transcription control region:• Core element: includes the transcription start site +1 and
The -31 to +6 sequence, which is essential for transcription.
• UCE: 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.
+1
Pre-rRNA gene
-100
Core element+6-31
UCE50-80 bp
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
• UBF• UBF-1 + UCE• UBF-2 + Part of core promoter• Two UBF interaction causing DNA to form a loop
3 TAF1 TBP
SL1
Upstream binding factor (UBF)
UCE core promoter UBF UBF
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
RNApol I
Allows RNA pol binding complex to initiate
Pre-rRNA
A low rate of basal transcription is seen in the absence of UBF, and this is greatly stimulated in the presence of UBF.
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
Selectivity factor 1SL1: An additional factor, called selectivity factor, it is essential for RNA pol I transcription. • SL1 binds to and stabilizes the UBF-DNA complex and
interacts with the free downstream part of the core element. • SL1 binding allows RNA pol I to bind to the complex and
initiate transcription, and is essential for rRNA transcription.
TBP and TAFIsSL1 has now been shown to contain several subunits, including:• TBP (TATA-binding protein). TBP is required for initiation by
all three eukaryotic RNA polymerases (see Ml, M3 and M5);• TAFIs: The other three subunits of SL1 are referred to as TBP-
associated factors or TAFs, and those subunits required for RNA pol I transcription are referred to as TAFIs.
3 TAF1 TBP
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
M3 RNA pol III genes: 5S and tRNA transcription
• RNA polymerases III• tRNA genes• 5S rRNA genes• Promoters of RNA pol. III• RNA Pol III termination
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
RNA pol. III
• Located in nucleoplasm
• With at least 16 or more subunits
• For the transcription for:
• 4s pre-tRNA;
• 5s pre-rRNA;
• snRNA (small nuclear RNA);
• Cytosolic RNA.
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
Transcription of tRNA genes• Cis-element including ( for pre-t RNA ) two highly conserved regions
--A box; TGGCNNAGTGG ( for coding D-loop of tRNA )--B box; GGTTCGANNCC ( for coding TC-loop of tRNA )
ACC
anticodon loop
5’
TC-loop acceptor arm
D loop
V loop
A box B box
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
TFIIIC
Fig. 1. Initiation of transcription at a eukaryotic tRNA promoter
A box B box
TFIIIC
B”TBP
BRF
RNA pol III
B”TBP
BRF
TFIIIB
RNA pol III
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
Initiation of transcription at a euk. tRNA promoter
TFIIIB binds 50 bp upstream from the A box. • TFIIIB consists of three subunits:
– TBP, the general initiation factor required by all three RNA polymerases;
– BRF (TFIIB-related factor), since this RNA pol II initiation factor has homology to TFIIB;
– B" factor. • TFIIIB has no sequence specificity and therefore its binding
site appears to be determined by the position of TFIIIC binding to the DNA.
• TFIIIB allows RNA pol III to bind and initiate transcription. Once TFIIIB has bound, TFIIIC can be removed without affecting transcription.
TFIIIC is therefore an assembly factor for the position of the initiation factor TFIIIB.
B”TBP
BRF
TFIIIB
TFIIIC
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
Transcription of 5S rRNA genes• 5S rRNA: RNA pol III transcribes the 5S rRNA of the
large ribosomal subunit. This is the only rRNA subunit to be transcribed separately.
• Tandem array: Like the other rRNA genes, the 5S rRNA genes are tandemly arranged in a gene cluster. In humans, there is a single cluster of around 2000 genes.
• C box: The promoters of 5S rRNA genes contain an internal control region called the C box which is located 81-99 bp downstream from the transcription start site.
• A box: A second sequence termed the A box around bases +50 to +65 is also important.
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
TFIIIC
Fig. 2. Initiation of transcription at a eukaryotic 5S rRNA promoter
TFIIICRNA pol III
B”TBP
BRF
TFIIIB
TFIIIA
A box C box
+81 +99+50 +65
TFIIIA RNA pol III B”TBP
BRF
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
Promoters of RNA pol. III
1. Eukaryotic tRNA promoter
2. Eukaryotic 5S rRNA promoter
A box B boxTATA box +1
-30 -23
A box C boxTATA box +1
-30 -23
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
RNA pol. III termination• Termination of transcription
by RNA pol III appears only to require polymerase recognition of a simple nucleotide sequence.
• This consists of clusters of dA residues whose termination efficiency is affected by surrounding sequence.
• For example, the sequence 5'-GCAAAAGC-3' is an efficient termination signal in the Xenopus somatic 5S rRNA gene.
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
M4 RNA pol II genes: Promoters and enhancers
• RNA polymerase II
• Promoters of RNA pol. II
• Upstream regulatory elements (URE)
• Enhancers
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
RNA polymerase II
• RNA pol II is located in the nucleoplasm. • It is responsible for the transcription of all
protein-coding genes (mRNA) and • some small nuclear RNA genes. • The pre-mRNAs must be processed after
synthesis, including:– cap formation at the 5'-end of the RNA and – poly(A) addition at the 3'-end, as well as – removal of introns by splicing.
Promoters of RNA pol. II TATA box / Hogness box / Goldberg-Hogness box (-30)
Rich AT and rich GC flanked (function=–10 in E. coli)
rich GC------T A T A ( A / T ) A ( A / T)------rich GC
82 97 93 85 63 (37) 83 50 (37)
1-4Kb -70 -30 -1C +1A
GC island CAAC box TATA box Cap
Enhancer UPE core promoter Promoter (basic factor)
-270 -170
URE
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
Upstream regulatory elementsThe low activity of basal promoters is greatly increased by
the presence of other upstream elements of promoter. URE: These sequences which are often located within
100-200 bp upstream from the promoter (-170~-270), and they are referred to as upstream regulatory elements (URE) and play an important role in ensuring transcri-ption from the promoter.
Two common examples are:• SP1box is found upstream of many genes both with and
without TATA boxes, and• CCAAT box. Promoters may have one, both or multiple copies of these
sequences.
Example: SV40 Enhancer (-179~ -250)
Enhancer
Enhance expression Basic expression
Position not be fixed isolated region
Bi-directional element Mono-directional element
Enhancer Promoter
No for special gene only for special gene
Enhancer vs. Promoter
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
M5 General transcription factors and pol. II initiation
• RNA Pol II basal transcription factors
• TFIID and TBP
• TFIIA, TFIIB and TFIIF
• Factors binding after RNA polymerase
• CTD phosphorylatiion by TFIIH
• TIC: Transcriptional Initiation Complex
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
RNA Pol II CTDF
RNA Pol IIF
CTD
RNA Pol II basal transcription factors• A series of nuclear transcription factors have been identified and purified. • These are required for the transcription initiation of RNA Pol II promoter. • These multisubunit factors are named TFIIA, TFIIB, etc. • They have been shown to assemble on basal promoters in a specific order.
A TBP
TAFIIs
ATFIIA
BB TFIIB
E
E
H
H
J
J
mRNA
TBP
TAFIIsTFIID
RNA Pol II CTDF
TATA box
TIC: (Transcriptional Initiation Complex)
Pre-TIC = Promoter + TFIID + TFIIA
• TBP: TATA-binding protein Needed for RNApol I, II, III (SL1, TFIIIB, TFIID) Very high conserved C-end domain of 180 aa Binds with DNA in minor groove & winds it
TFIID: A sort of protein complex (TBP & > 8 TAF)
TFIID and TFIIA
TFIIA: Binds to TFII D
Enhances TFII D binding to TATA box
Stabilizing the DNA-TFII D complex
TATA +1 Winds minor groove
TFII A
TFII D pre-TIC
1×3
TFIIB and TFIIF
TFIIB: Once TFIID has bound to DNA, TFIIB binds to TFIID and binds to TBP of TFIID and RNA pol. II This seems to be; TFIIB as a bridging factor allowing recruitment of RNApol II to TIC together with TFIIF TFIIF: Including two subunits of RAP30, RAP74 Binding and recruiting RNApol II to assemble TIC Promoting RNA elongation by its helicase (ATPase)
Basic TIC = Pre-TIC + TFIIB + [ RNA pol. II + TFIIF ]
TFII F RNApol IITFII B
Basic TIC
2×3
TFIIH: Large complex made up of > 5 subunits Helicase activity (ATPase) & kinase activity Phosphorylation of CTD of RNApol II DNA repair TFIIE: Associate with TFIIH kinase
Factors binding after RNA polymerase
TFIIJ: remains to be fully characterized.
Complete TIC = Basic TIC + TFIIH + TFIIE + TFIIJ
complete TIC E
H J
3×3
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
TATA +1 Winds minor groove
TFII A TFII D
Pre -TIC
TFII F RNApol IITFII B
Basic TIC
conclusion
TIC: Transcriptional Initiation Complex
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
mRNA
Promoter clearance
Transcription starting
complete TIC
E
HJ
E H B D AJ
9 - 6 = 3
--L’ max. subunit is 240 kd & has specific COOH-end
named CTD ( Carboxyl Terminal Domain )
only in RNApol II
--CTD is essential for viability of enzyme;
The CTD of RNA pol. II
Yeast 26XDrosophila 44X repeat unit of 7 aa / in CTDRat & Human 52X
IIO (240kd) IIA (220kd) IIB (180kd)
With all Without all Basic form
RNApol easy to leaves Promoter for elongation
--CTD-end construction:
7aa repeats & high frequency phosphorylation
Tyr—SerP—Pro—ThrP—SerP—Pro—SerP
Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences
That’s all for Section M