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L10
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5
-GATCCA-3
Direction of DNA polymerase III on this strand fragment (rightor left) ? DNA synthesis complementary making and moving.
What is the sequence of the leading template strand ?Base pair complementWhat is the sequence of the corresponding Okazaki fragment ?
Complementary strand directions
Replisome structure and
accessory proteins arehighly conserved, fromvirus to eukaryotes
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RNA is similar to DNA, but,1. RNA is usually single stranded,
2.
The sugar-phosphate backbone is
composed of ribose sugar, (2
OH),not deoxyribose sugars
3.
RNA contains the pyrimidine base
URACIL instead of thymine.
4.
RNA can catalyze biological
reactions
3
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RNA has a
sugar-phosphate backbone(phosphodiester bonds).
The ribose has a OH on
the 2
carbon.
RNA is single stranded.
RNA is has 5
to 3
polarity like DNA.
4
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There are two grades and 6+ classes of RNA: pp 287
(1) Gene information
genes (a)Messenger mRNAis the protein encoding transcript of agene(90% of known genes, 1% of RNA in humans)
(2) Functional RNA-may have catalytic propertiesgenes ? (b)Ribosomal RNA rRNA is part of the translation
complex.
genes ? (c) Transfer RNA tRNA participates in translation. Itcarries specific amino acids to be incorporated into the new protein.
genes? (d) Small nuclear RNAs(sn RNAs)- spliceosome, rRNA
assembly specific to eukaryotes.
genes? (e)Micro RNA
s - short 20-22 nucleotide bases, single
stranded RNA
s that may be involved in gene expression or mayblock the translation of mRNA.
genes ?(f)Small interfering RNAs (siRNA) and piwi- interacting
RNAs (piRNA) - anti virus and transposable element
genes ? (g)Long noncoding RNA (lncRNA)) - transcriptional control
(XIST) and epigenetic regulation (200 + bases.
5
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RNA has (1) sugar-phosphate backbone
(2) 4 nucleotides (A U, C, G)
(3) directionalityIt is synthesized from the 5
end to the 3
end.
But, it is single stranded and has uracil instead of thyamine
Convention - DNA is always drawn with the upper strandrepresented in the 5 to 3 direction, mRNA same .
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DNA is double stranded.
5-GCACTACGCATCGATCGACTAGCTAGCATC-33-CGTGATGCTTAGCTAGCTGATCGATGCTAG-5
The standard representation is the non template orcoding strand:
GCACTACGCATCGATCGACTAGCTAGCATC
Assume the sequencerepresents double stranded DNA
with the upper strand shown, in the 5 to 3 direction,
(unless text describes it otherwise).
7
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DNA
RNA
mRNA is transcribed from DNA 5- 3 in the coding strand order
-The RNA producthas the same sequence as the upper, coding
strand of DNA,except in has Us in place of Ts,BUT
The lower strand of DNA is the physical template for RNA synthesis.
RNA is drawn in the 5
(left) to 3
(right) direction.
8
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The 8thedition refers to the coding and template strands.
The 9th & 10thedition (white& black books ) refers to template and
non- template strands, which is called the coding strand(pp 289).
There is not always consistency between books or people
, be careful, check sequence and directionElsewhere you may see:
Template strand = sometimes coding strand, nonsense strand,
antisense strand
Non template strand =coding, non coding strand,sense strand
9VARIATION IN NOMENCLATURE
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Gene function - TRANSCRIPTION
Gene transcription the primary transcript or pre
mRNA is synthesized or transcribed from the DNA
template 5
- 3
and may then be modified into the
message (mRNA)
transcription translation
DNA mRNA Protein (central dogma)
10
But we already know of 1 exception .
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There are 3 stages of transcription:
Initiation, Elongation, Termination
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12
A Promoteris a (small) region of (consensus) sequenceelements of DNA, which are necessary to initiatetranscription.
Conserved sequence-if several nucleotide sequences (amongspecies) align perfectly or close to it, - the same nucleotidesequence.
Consensus sequence-if several sequences align but not soperfectly- there is some variation among sequences, but asignificant percentage of nucleotides co-occur at a highfrequency.
Initiation - Bacterial transcription is initiated (1) at apromoter sequence (2) when a RNA polymerase holoenzyme
binds to it.
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Promoter sequences and the consensus sequence(promoters or
promoter elements)are on the 5
side (upstream) of of the
transcription start site (1+) coding strand
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RNA polymerase is a multimericprotein complex .
E. coliRNA polymerase.
Prokaryote Initiation
-assembly of core enzyme, interactions with regulatory proteins
- catalysis -ribonucleoside triphosphate binding site
- binds to DNA template, helicase activity
!- core enzyme assembly, regulation of gene expression
#- binding core enzyme to the promoter(position holoenzyme -10,-35),
strand separation,
Holoenzyme
(complete
enzyme)
core enzyme
14
One of
many
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15
5
UTR(untranslated
region)
one of man
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Eukaryotic Initiation
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Eukaryotic DNA is more complicated than Prokaryotic
(1)Many more genes,
(2)
more DNA and
(3)there is more intervening (non message-coding) DNA in eukaryotes,
thus the gene density is lower in eukaryotes:1/1400 bp in E. coli
1 gene /9000 bp in Drosophila,
1 gene / 100,000 bp in humans
(4) chromatin structure plays a key role in gene transcription.
(5) more complicated cellular structureTHUS , it is not surprising thatRNA polymerases are more
complicated in eukaryotes, requiring more polymerases, a more
complex promoter, accessory and regulatory proteins (transcription
factors)
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In eukaryotic organisms:
-RNA polymerase Itranscribes rRNA
in the nucleolus, except for the small
5S rRNA (large fraction of
transcription).
-RNA pol II transcribes all protein
coding genes, some snRNA
s, in thenucleus,LncRNA.
-RNA pol IIItranscribes small
functional RNA genes such as those
in the spliceosome, 5S rRNA, transferRNA (tRNA),sn RNA
s not made by
RNA pol II in the nucleus
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(1) Core promoterEukaryotic genes have a TATA (TATAAAA) box isabout -30 region and an initiator site which spans 1+, specifying
where the transcription polymerase assembles and beginsOther
promoter sites: -40 and -120 (GC), -80 (CAAT), -120 (Octamer)
(2) (a) There are many additional cis - regulatorysequences (activators100s bp + upstream only, enhancers ~ 1000 bp +, up and downstream)
(b) trans actingGeneral Transcription Factors (GTFs), and
(c) in vertebrates, particularly mammals, the absence of histone
methylation (nucleosomes)near the promoter allows expression.
Eukaryotic Initiation 19
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Figure 4-57 Molecular Biology of the Cell( Garland Science 2008)
(1) Expression starts withunwinding DNA, starting withthe nucleosome, although it is inan extended form in G1, early G2
18
Extended form, local unwindingof nucleosomes
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(1) Remove methyl tags and unwind nucleosomes
(2) TranscriptionBindingProteinat the TATA box - attracts other
GTFs (TBP is part of 1, of several GeneralTranscriptionFactors)+ RNA polymerase II core, forming thepre-initiation complex(3) Interaction of (upstream) cis-enhancer sequences
(4) Transcription Initiation
(5) Dissociation of GTP and Elongation
TBP
20Nucleosome wound, promoter methylated
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Transcription initiation in eukaryotes 21
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Transcription initiation in eukaryotes
TBP is part of TFII D protein (and several TBP associated factors)TFIID is one of several GTFs -general transcription factors
or Transcription Factor for RNA polymeraseII X(X=factor letter)
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Transcription initiation in eukaryotes
TBP attracts other GTF
s and then,
the RNApolymerase II coretogether the preinitiation complex
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Transcription initiation in eukaryotes
Transcription is initiated with the
phosphorlation of the Carboxyl
TailDomain,RNA polymerase
dissociates from most of the
GTF
s, but some remain at the
promoter-attracting the next core
enzyme
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The bases in RNA are added in a sequence that is complementary
to the DNA sequence G opposite C
s
C opposite G
s
U opposite A
s
A opposite T
s
ELONGATION in General 25
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RNA polymerase opens the DNA duplex, RNA is synthesized in the
5
to 3
direction from one strand of DNA and then closes it. A single
gene is only transcribed in one direction.
Only one strand is the template for 1 geneChromosomes have different genes in different orientations, so
different strands may be transcribed for different genes at
different locations.26
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27
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There are transcription termination signals in the DNA, beyond theprotein coding sequence:
(1)Intrinsic- GC rich hairpin which disrupts DNA-RNA binding
(2)Rho(a helicase) dependent binding site (rut=rho utilization site),
rho unwinds RNA&DNA facilitating RNA polymerase release.
ProkaryoteRNA transcriptiontermination28
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Cotranscriptional processing of RNA: capping
The initial RNA transcript is capped with a 7-methylguanosinetriphosphate.
The linkage is 5 to 5 and the three phosphates are maintained,
unlike RNA (or DNA) synthesis catalyzed by RNA polymerase.
Carboxyl TailDomain
30
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Cotranscriptional processing of Eukaryotic RNA
2. Most eukaryotic genes have
blocks of coding(exons) and noncoding (intron) DNA
The mRNA is transcribed primary
transcript(pre mRNA) with the
introns and the exons andthe introns are spliced outbefore
the mRNA is translated.
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Many human genes have alternate splicing patterns several
different related proteins can be produced by one gene.
33products of several loci can also be spliced into one mRNA.
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Cotranscriptional processing of RNA
Termination: when the highly
conserved sequenceAAUAAA or
AUUAAA is recognized , it signals a
termination enzyme to cut the end
~20 bases downstream and add a
polyA tail (AAA)- polyadenylation
signal
capping
splicing
34
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The mRNA is cleaved about 20 bp after a polyadenylation signal
and a poly (A) tail of about 300 nucleotides is added to the 3
end
of the mRNA.
2. 35Coding RNA
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Box 1. Key Genetic Features of Multicellular Organisms - S. B.
Caroll (2005) Evolution at 2 levels Plos Biology
Individual regulatory proteins function in many different contexts.
The expression of individual genes is multiply regulated, tissue-specific
and temporal controlled.
Many regulatory proteins are members of large families and can overlap
in function..
Multiple protein forms may be encoded by single genetic loci.Alternative
protein forms (isoforms) may function in different contexts and/or
possess different activities.