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Berg • Tymoczko • Stryer
Biochemistry Sixth Edition Chapter 29 RNA Synthesis and Processing Part I: RNA synthesis Copyright © 2007 by W. H. Freeman and Company
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RNA synthesis is a key step in the expression of genetic information Christmas tree: Active transcription
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RNA synthesis, or transcription, is catalyzed
by RNA polymerase Structural conservation Mg2+ ion (more complex regulation)
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RNA synthesis (or biological polymerization): initiation, elongation, and termination RNA polymerase’s functions: Search “promoters” (initiation sites; or cis-acting elements) Unwind short stretch of dsDNA Formation of phophodiester bonds (using rNTP) processivity Detect termination signals Interact with activator and repressor (transcription factors or trans-acting factors) regulation of txn rate
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RNA synthesis for mRNA, tRNA, and rRNA:
Common in chemistry and steps Different: RNA polymerase, regulation, and post-transcriptional processing
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Transcription in E coli
(holoenzyme) Core enzyme (contains catalytic site) s: finds promoter, initiates txn, then dissociates
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RNA polymerase active sites:
Similar to DNA pol. but overall structure is dif.
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Transcription initiation:
Identification of promoter sites footprinting technique
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Two common motifs are present on the 5’ (upstream)
side of the transcription start site -35 sequence and -10 sequence (consensus seq.) Core promoter
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Two common motifs are present on the 5’ (upstream)
side of the transcription start site -35 sequence and -10 sequence (consensus seq.) Core promoter Start site = +1 (+2…, -1, -2, etc.) Template strand vs. coding strand antisense (-) sense (+)
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Transcription initiation:
promoter activity (or efficiency) 1. strong promoter vs. weak promoter (in relation to consensus seq.) 2. Distance between two conserved seq. Other factors: * Promoter-binding proteins, or polymerase-binding proteins * UP element (upstream): -40~-60 of highly expressed genes (for a subunit)
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Transcription initiation: recognition of promoter sites s subunit
Help RNA pol. bind core promoter Transcription initiation: recognition of promoter sites s subunit
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Transcription initiation:
Search for promoter sites * Holoenzyme slides along dsDNA * Promoter is encountered by a random walk in one dimension * s subunit dissociates when nascent RNA chain = 9-10 nt * s subunit next assists another initiation
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There are multiple types of s subunit
s32: recognize heat-shock genes s54: responds to nitrogen starvation s determines where txn starts
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Transcription initiation: Template unwinding
Closed promoter complex open promoter complex: Important step in txn initiation
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Negative supercoiling facilitates unwinding
Exception: promoter of Topoisomerase II gene negative supercoils decreases txn (negative feedback) Supercoils: change structual relation of the –10 and –35 regions
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RNA synthesis can start de novo
First base at 5’end: pppG or pppA
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RNA synthesis grow in the 5’-3’ direction
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Transcription elongation: Txn bubble on DNA template
Elongation: 1. formation of first bond 2. dissociation of s strong binding of core 3. 50 nt/sec txn bubble 8bp
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Surface model of a bubble
“melted” DNA Surface model of a bubble
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RNA-DNA hybrid separation by RNA pol. Fixed structure of the txn bubble
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Transcription elongation:
Fidelity of the RNA pol. Contains proofreading nuclease activity 104 – 105 (DNA polymerase: 107) Higher error rate can be tolerated: *Mistakes are not transmitted *Many RNA transcripts for most genes
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Transcription termination:
Formation of phosphodiester bond stops RNA-DNA hybrid dissociates Melted DNA rewinds RNA pol. releases DNA Transcribed region of DNA contains top signal
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Palindromic GC-rich region followed by AT-rich
In DNA: Palindromic GC-rich region followed by AT-rich Stable hairpin because of GC-rich Txn stop
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How does this structure terminate transcription?
RNA pol. pauses after such structure rU-dA: highly unstable Weakly bound nascent RNA dissociates
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Transcription termination:
Additional factor, rho (r) How does r termination of RNA synthesis?
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72 nt Hexameric rho is ATPase in the presence of ssRNA
Activated by C-rich & G-poor RNA region Hydrolysis RNA-DNA helicase: breaking hybrid helix 72 nt
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Transcription termination:
Additional factor, nusA protein binds specific termination signals in E. coli: “Attenuators” Important feature of the termination mechanism: signals lie in RNA (not DNA)
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Antibiotics: inhibitors of transcription
Example no. 1
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Rifampicin blocks txn initiation
Binds a pocket occupied by newly formed DNA-RNA hybrid Competitor Conserved in prok. not in euk. antibiotic
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Example no. 2: actinomycin D
* Binds specifically and tightly to dsDNA * Intercalation: between neighboring base-pair * at low concentrations, inhibit txn but not DNA replication (both prok. and euk.)
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Eukaryotic transcription: more complex regulation
Differential txn regulation cell types
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Three keys in euk.: Nuclear membrane Txn regulation: more promoters, enhancer RNA processing: ex. splicing
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Three different types of euk. RNA polymerases
* Large proteins * 8-14 subunits Similarity: RNA pol. II: 220-kd (largest) subunit has a carboxyl-terminal domain (CTD) YSPTSPS repeats serine phosphorylation
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Binds to pol. II tightly and inhibits elongation
(action is conserved in euk.)
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Euk. transcriptional initiation: promoters
Conserved sequences for pol. binding Different in sequence and position RNA polymerase-dependent
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Promoter binding proteins
RNA polymerase I: rDNA gene X hundreds TATA-like seq.: Ribosomal initiator element Promoter binding proteins RNA pol. I
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RNA polymerase II a set of conserved elements: combination
Enhancer: unique to euk., more than 1 kb from promoter
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RNA polymerase III: intergenic
(within transcribed region)
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RNA polymerase II promoter: 3 common elements
Mutagenesis exp’ts, footprinting, and sequence comparisons 1 2 Initiator element (-3 ~ +5) 3 Downstream core promoter element (+28 ~ +32)
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-30 ~ -100 (similar to prok. –10)
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On template (antisense) strand
-40 ~ -150 (similar to prok. –35) On template (antisense) strand Constitutively expressed genes Euk. promoter elements: recognized by txn factors
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Txn initiation: TFII’s TFIID to TATA box (seq. specific)
Basal txn apparatus Binds “mediator”
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TBP and TATA-box DNA * Unwinding (minor groove)
* Hydrophobic interaction * Phenylalanine intercalation * Asymmetry
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initation elongation
(by TFIIH) initation elongation * Stabilize txn elongation * Recruit RNA-processing enzymes
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Multiple transcription factors bind euk. promoters
* Basal txn apparatus: minimal transcription (low) * Additional txn factors: bind other sites for high txn rate * Upstream stimulatory sites: variable (sequence & location)
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There are many transcription factors
Ex. heat-shock transcription factor (HSTF) bind directly to consensus seq. in response to high temperature
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Another level of promoter activity: enhancers
* Have no promoter activity * Act from several thousand bp away * Can be downstream, upstream, or intragenic * Cell type-specific In yeast: upstream activator sequences (UASs) txn factors and cis-acting elements: key to txn regulation transcription: key to gene expression
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