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

BiochemistrySixth Edition

Chapter 29RNA Synthesis and Processing

Part I: RNA synthesis

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

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

(more complex regulation)

Mg2+ ion

RNA synthesis (or biological polymerization):initiation, elongation, and termination

RNA polymerase’s functions:1. Search “promoters” (initiation sites;

or cis-acting elements)2. Unwind short stretch of dsDNA3. Formation of phophodiester bonds

(using rNTP) processivity4. Detect termination signals5. 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)

: 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

Core promoter

(consensus seq.)

Two common motifs are present on the 5’ (upstream) side of the transcription start site

-35 sequence and -10 sequence

Core promoter

(consensus seq.)

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 subunit)

Transcription initiation:recognition of promoter sites subunit

Help RNA pol. bindcore promoter

Transcription initiation:Search for promoter sites

* Holoenzyme slides along dsDNA* Promoter is encountered by a random

walk in one dimension* subunit dissociates when nascent RNA

chain = 9-10 nt* subunit next assists another initiation

There are multiple types of subunit

70

32: recognize heat-shock genes54: responds to nitrogen starvation

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 strong binding of core 3. 50 nt/sec

txn bubble

8bp

Surface model of a bubble

“melted” DNA

RNA-DNA hybrid separation by RNA pol.

Fixed structure of the txn bubble

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 elongation:

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

In DNA:Palindromic GC-rich regionfollowed by AT-rich

Stable hairpin because of GC-rich

Txn stop

How does this structure terminate transcription?

1. RNA pol. pauses after such structure2. rU-dA: highly unstable3. Weakly bound nascent RNA dissociates

Transcription termination:Additional factor, rho ()

How does termination of RNA synthesis?

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.:1. Nuclear membrane2. Txn regulation:

more promoters,enhancer

3. 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

1. Conserved sequences for pol. binding2. Different in sequence and position3. RNA polymerase-dependent

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

3

Initiator element (-3 ~ +5)

Downstream core promoter element (+28 ~ +32)

-30 ~ -100(similar to prok. –10)

-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

(by TFIIH)

initation elongation

* Stabilize txn elongation* Recruit RNA-processing enzymes

* Basal txn apparatus: minimal transcription (low)

* Additional txn factors: bind other sites for high txn rate

* Upstream stimulatory sites: variable (sequence & location)

Multiple transcription factors bind euk. promoters

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 regulationtranscription: key to gene expression