transcription. the central dogma dna mrna protein transcription translation gene expression rpe65...
TRANSCRIPT
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Transcription
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the Central Dogma
DNA
mRNA
Protein
transcription
translation
geneexpression
RPE65 gene
RPE65 protein
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Transcription Individual DNA regions (genes) copied to mRNA One DNA strand is template Single-stranded RNA produced
template strand
mRNA
template strand
template strand
template strand
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Transcription Overview
Un beau jour, je suis allé au marché pour acheter du pain. Il faisait chaud. Alors, j’ai acheté aussi un
limonade.
Il faisait chaud.
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mRNA
DNA
transcription
CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC
CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGCGATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG
gene
• What do we call this strand?
Transcription overview
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mRNA
DNA
transcription
CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC
CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGCGATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG
template strand
• What enzyme makes RNA?
Transcription overview
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mRNA
DNA
transcription – RNA polymerase
CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC
CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGCGATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG
template strand
• What direction is mRNA made?
Transcription overview
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mRNA
DNA
transcription – RNA polymerase
CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 3’5’
CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGCGATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG
template strand
• What direction is the template strand read?
Transcription overview
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mRNA
DNA
transcription – RNA polymerase
CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 3’5’
CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGCGATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG
• Which strand does the mRNA look like?
5’3’
Transcription overview
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mRNA
DNA
transcription – RNA polymerase
CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 3’5’
CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGCGATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG
• How do we know where to start and stop?
5’3’
Transcription overview
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Transcription overview RNA polymerase synthesizes RNA 5′→ 3′ Starts at promoter, ends at terminator
promoter terminator
+1
5′ UTR
coding region
startcodon
stopcodon
“upstream” “downstream”
DNA
transcription
coding region
startcodon
stopcodon
3′ UTR
5′ 3′mRNA
translation
NH3 COOHprotein
• How is the RNA actually made?
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Prokaryotic transcriptionPromoter: -10 and -35 sequences
DNA
-35-35 -10-10 +1+1mRNA
5′ TTGACAT AACTGTA
5′ TATAAT ATATTA
5’3’
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Prokaryotic transcriptionPromoter: -10 and -35 sequences
DNA
-35-35 -10-10 +1+1mRNA
TTGACAT TATAAT
5’3’
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Prokaryotic transcriptionInitiation:
RNAP sigma subunit (σ) binds -10 and -35
DNA
-35-35 -10-10 +1+1σ5’
3’
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Prokaryotic transcriptionInitiation:
RNAP core (α2ββ’) binds sigma
DNA
-35-35 -10-10 +1+1σ
α2 ββ’“core”5’
3’
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Prokaryotic transcriptionInitiation:
Promoter determines template strand and direction
-35-3555′′ 33′′33′′ 55′′
-10-10
-35 -35-10 -10
template strandfor gene 1
template strandfor gene 2
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Regulatory elements Prokaryotes use operator sequences
DNA
-35-35 -10-10 +1+1mRNA
TTGACAT TATAAT
5’3’
OperatorsProtein Transcription factors
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Prokaryotic transcriptionInitiation:
RNAP opens transcription bubble (helicase activity)
DNA
-35-35 -10-10σ
+1+1
5’3’
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Prokaryotic transcriptionInitiation:
RNAP begins mRNA synthesis at +1
DNA
-35-35 -10-10σ
+1+1
5’3’ mRNA
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Prokaryotic transcriptionInitiation:
Sigma released
DNA
-35-35 -10-10σ
+1+1
5’3’ mRNA
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Elongation:
Prokaryotic transcription
DNA
-35-35 -10-10
5’3’
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Elongation:
Prokaryotic transcription
DNA5’3’
terminator
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Replication Transcription Synthesize DNA Copy whole genome Copy both strands Need primer 5′ → 3′ Multiple enzymes
• How are replication and transcription similar?
• How are they different?
Synthesize RNA Copy one gene Copy one strand No primer 5′ → 3′ Only RNA polymerase
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Eukaryotic transcription 3 RNA polymerases:
RNA polymerase I – rRNA RNA polymerase II – mRNA RNA polymerase III – tRNA
RNA polymerase IIfrom yeast
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Eukaryotic transcription RNAP II recognizes:
TFIID bound to TATA box (TATAAA) TFIIB bound to TFIID Transcription factors bound to enhancer sequences
+1
Enhancers
Transcription factors
Sp1 hERR1 CAAT GATA TATAbox
TFIIB TFIID
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Eukaryotic transcription RNAP II recognizes:
TFIID bound to TATA box (TATAAA) TFIIB bound to TFIID Transcription factors bound to enhancer sequences
+1
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Different from Prokaryotes- No terminator! RNA cleaved from transcription complex
+1
AAUAAA
AAUAAA
Eukaryotic Transcription Termination
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RNA processing in eukaryotes
DNA
promoter
exons introns primary transcript(nucleus)
5’ capAAAAAAAAA3’ poly-A tail
AAAAAAAAA
splicingsplicing
transcriptiontranscription
unbroken coding sequence
transport to cytoplasm for translationtransport to cytoplasm for translation
final mRNA
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methylated guanine “backward” 5′ to 5′ linkage Not encoded in DNA Capping enzyme Recognition by ribosome
5′ cap
5′ AGACCUGACCAUACC
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RNA processing in eukaryotes
DNA
promoter
exons introns primary transcript(nucleus)
5’ capAAAAAAAAA3’ poly-A tail
AAAAAAAAA
splicingsplicing
transcriptiontranscription
unbroken coding sequence
transport to cytoplasm for translationtransport to cytoplasm for translation
final mRNA
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3′ poly(A) tail Poly(A) polymerase Add ~200 A’s Not in template Important for:
Export of mRNA Initiation of Translation Stability of mRNA
…UGGCAGACCUGACCA 3′
…UGGCAGACCUGACCAAAAAAAAAAAAAAAAAAAA
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RNA processing in eukaryotes
DNA
promoter
exons introns primary transcript(nucleus)
5’ capAAAAAAAAA3’ poly-A tail
AAAAAAAAA
splicingsplicing
transcriptiontranscription
unbroken coding sequence
transport to cytoplasm for translationtransport to cytoplasm for translation
final mRNA
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Splicing Most genes interrupted by introns Introns removed after transcription Exons spliced together
5’ capAAAAAAAAA3’ poly-A tail
AAAAAAAAA
splicingsplicing
unbroken coding sequencefinal mRNA
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Splicing snRNPs recognize exon-intron
boundaries RNA + protein Cut and rejoin mRNA
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Splicing
RPE65 mRNA in nucleus: 21,000 nt (14 exons)
AAAAAAAAA
AAAAAAAAA
splicingsplicing
mature RPE65 mRNA in nucleus: 1,700 nt (8%)
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Splicing Alternative splicing: >1 protein from one gene 27,000 human genes, but >100,000 proteins
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Splicing
Mutations affecting splicing can cause genetic disease:cystic fibrosis retinitis pigmentosaspinal muscular atrophy Prader-Willi syndromeHuntington disease spinocerebellar ataxiamyotonic dystrophy Fragile-X syndrome
Or produce genetic susceptibility to disease:lupus bipolar disorderschizophrenia myocardial infarctiontype I diabetes asthmacardiac hypertrophy multiple sclerosisautoimmune diseases elevated cholesterol