Berg • Tymoczko • Stryer Biochemistry Sixth Edition Chapter 29 RNA Synthesis and Processing Part I: RNA synthesis Copyright © 2007 by W. H. Freeman and Company

RNA synthesis is a key step in the expression of genetic information Christmas tree: Active transcription

RNA synthesis, or transcription, is catalyzed by RNA polymerase Structural conservation Mg2+ ion (more complex regulation)

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

RNA synthesis for mRNA, tRNA, and rRNA: Common in chemistry and steps Different: RNA polymerase, regulation, and post-transcriptional processing

Transcription in E coli (holoenzyme) Core enzyme (contains catalytic site) s: finds promoter, initiates txn, then dissociates

RNA polymerase active sites: Similar to DNA pol. but overall structure is dif.

Transcription initiation: Identification of promoter sites footprinting technique

Two common motifs are present on the 5’ (upstream) side of the transcription start site -35 sequence and -10 sequence (consensus seq.) Core promoter

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 (+)

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)

Transcription initiation: recognition of promoter sites  s subunit Help RNA pol. bind core promoter Transcription initiation: recognition of promoter sites  s subunit

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

There are multiple types of s subunit s32: recognize heat-shock genes s54: responds to nitrogen starvation  s determines where txn starts

Transcription initiation: Template unwinding Closed promoter complex  open promoter complex: Important step in txn initiation

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

RNA synthesis can start de novo First base at 5’end: pppG or pppA

RNA synthesis grow in the 5’-3’ direction

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

Surface model of a bubble “melted” DNA Surface model of a bubble

RNA-DNA hybrid separation by RNA pol. Fixed structure of the txn bubble

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

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

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

How does this structure terminate transcription? RNA pol. pauses after such structure rU-dA: highly unstable Weakly bound nascent RNA dissociates

Transcription termination: Additional factor, rho (r) How does r termination of RNA synthesis?

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

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)

Antibiotics: inhibitors of transcription Example no. 1

Rifampicin blocks txn initiation Binds a pocket occupied by newly formed DNA-RNA hybrid  Competitor Conserved in prok. not in euk.  antibiotic

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.)

Eukaryotic transcription: more complex regulation Differential txn regulation  cell types

Three keys in euk.: Nuclear membrane Txn regulation: more promoters, enhancer RNA processing: ex. splicing

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

Binds to pol. II tightly and inhibits elongation (action is conserved in euk.)

Euk. transcriptional initiation: promoters Conserved sequences for pol. binding Different in sequence and position RNA polymerase-dependent

Promoter binding proteins RNA polymerase I: rDNA gene X hundreds TATA-like seq.: Ribosomal initiator element Promoter binding proteins RNA pol. I

RNA polymerase II a set of conserved elements: combination Enhancer: unique to euk., more than 1 kb from promoter

RNA polymerase III: intergenic (within transcribed region)

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)

-30 ~ -100 (similar to prok. –10)

On template (antisense) strand -40 ~ -150 (similar to prok. –35) On template (antisense) strand  Constitutively expressed genes Euk. promoter elements: recognized by txn factors

Txn initiation: TFII’s TFIID to TATA box (seq. specific) Basal txn apparatus Binds “mediator”

TBP and TATA-box DNA * Unwinding (minor groove) * Hydrophobic interaction * Phenylalanine intercalation * Asymmetry

initation  elongation (by TFIIH) initation  elongation * Stabilize txn elongation * Recruit RNA-processing enzymes

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)

There are many transcription factors Ex. heat-shock transcription factor (HSTF) bind directly to consensus seq. in response to high temperature

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