Welcome to Chapter 12 Mechanisms of transcription.

Welcome to Chapter 12

Mechanisms of transcription

Introduction Up to this point we have been

considering maintenance to the genome ,that is ,how the genetic material is organized ,protected, and replicated.

In the next parts ,we will describe the basic processed responsible for gene expression.

First let us go into the world of transcription

Transcription Vs Replication

Transcription is chemically and enzymatically, very

similar to DNA replication.Both involve

enzymes that synthesize a new strand of nucleic acid

complementary to DNA template

strand.Moreover ,there are many differences between

them.

The differences go as follows: RNA is made of ribonucleotides RNA polymerase ,which catalyzes the

reaction,needs no primer The newly synthesized RNA does not remain

base-paired to the template DNA strand Less accurate ,one mistake occurs in 10,000 Because of different purpose ,transcription

selectively copies only certain parts of the genome and makes anything from one to several hundred,or even thousand.

Question :why transcription is less accurate than replication?

I think the difference makes good sense if we associate it with the results of the mistake

s.DNA is the molecule in which the genetic material is stored,and DNA replication si the process by which that genetic material is passed on.Any mistake can easily be catastrophic:it becomes permanent in the genome of that individual and also gets passed on to su

bsequent generations.

Transcription ,in contrast,produces only transient copies and normally several from each transcribed region.

Thus ,a mistake during transcription will rarely do more harm than render one out of many transient transcripts defective.

Outline

1. RNA polymerase & Transcription cycle

2. The transcription cycle in bacteria

3.Transcription in eukaryotes

Topic 1:

RNA polymerase & The transcription cycle

RNA polymerase RNA pol come in different

forms ,but share many features,especially in those parts of the enzyme directly involved with catalyzing the synthesis of RNA

RNA pol performs essentially the same reaction in all cells,from bacteria to humans.

1 The structure of RNA pol From bacteria to mammals ,the cellular

RNA polymerase are made up of multiple subunits .

Bacteria have only a single RNA pol ,which is the core enzyme capable of synthesizing RNA

Eukaryotic cells have three, namely RNA pol I ,II ,and III .They are responsible for synthesis of different kinds of RNA

Table 12-1: The subunits of RNA polymerases

“Crab claw” shape of RNAP (The shape of DNA pol is__)

Active center cleft

’

RPB3

RPB11

RPB2

RPB1

RPB6

Fig 12-2 RNAP Comparison

The same color indicate the homologous of the two enzymes

prokaryotic

eukaryotic

RNA pol II is the focus ,which is responsible for transcribing most genes-indeed,essentially all protein-encoding genes.

RNA Pol I transcribes the large ribosomal RNA precursor gene.

RNA Pol III transcribes tRNA genes,some small nuclear RNA genes,and the 5S rRNA gene

Since the structure of RNA Pol is this,there come the question:How do they

function? Or how do they realize the process of

transcription?

Transcription by RNA Pol proceeds in a series of steps

Initiation

Elongation

Termination

Let us go deep into the details

Process 1: Initiation(1)Promoter :the DNA sequence that

initially binds the RNA pol(2)Promoter-polymerase complex

undergoes structural changes(3)The DNA around the point where

transcription unwinds,forming a “bubble”( similar to DNA replication)

(4)Again like DNA replication,the direction of transcription is from 5’ to

3’

Additionally ,unlike replication,only one of the DNA strands acts as a template on which the RNA strand is built.

Transcription Initiation Invoves 3 Defined Steps

Form closed complex Form open complex Form stable ternaty complex

Fig 12-3-initiation

Binding (closed complex)

Promoter “melting” (open complex)

Initial transcription

Closed complex

Initial binding of pol to a promoter

In this form ,DNA remains double-stranded,and the enzyme is bound to one face of the helix.

Open complex

DNA strands separate around the transcription site

The transcription bubble forms

Stable ternary complex Enzyme escape the promoter once it g

ets further than the 10 bp Stable ternary complex contains enzy

me,DNA and RNA Then the elongation phase comes

Process 2 : Elongation Begins when the enzyme has synthesized a

short stretch of RNA (about 10 bp) The RNA pol undergoes further comformati

onal changes to grip the template more firmly

The enzyme functions:RNA synthesis ,unwind the DNA chains in front,re-anneal it behind,dissociate the growing RNA chain from the template

Fig 12-3-Elongation and termination

Termination

Elongation

Process 3: Termination Once the length of the gene has

been transcribed ,the RNA pol must stop and release the product

In some cells ,there are specific,well-characterized sequences.In other cells,it remains to be seen what instructs the termination

Topic 2 :The Transcription Cycle In Bacteria

2-1 Bacterial promoters vary in strength & sequence,but have certain defining features

The bacterial core RNA pol can ,in principle ,initiate transcription at any point on a DNA molecule .In cells,polymerase initiates transcription only at promoters.

It is the addition of initiation factor called σthat converts core enzyme into the form that initiates only at promoters.

That form of the enzyme is called holoenzyme ,which is made up of core enzyme and σfactor

Fig 12-5a: bacterial promoterThe distance is conserved

1. 70 promoters contain recognizable –35 and –10 regions, but the sequences are not identical.

2. Comparison of many different promoters derives the consensus sequences reflecting preferred –10 and –35 regions

The details of σ factor Structure : composed of 4 regions

called σregion 1 through σregion 4 Function :recognize the site of

promoter, mediates binding of polymerase to the promoter

Fig 12-6: regions of

Region 4 recognizes -35 element Region 2 recognizes -10 element

Region 3 recognizes the extended –10 element

Figure 12-4

,

Holoenzyme= Holoenzyme= factorfactor + + core enzymecore enzyme

In cell, RNA polymerase initiates transcription only at promoters. Who confers the polymerase binding specificity?

Take E.coli as a exampleIn the case of E.coli ,the predominant σfact

or is calledσ70 factor .Promoters recognized by σ70 factor share the following characteristic structure:two conserved sequences,each of six nucleotides,are separated by a nonspecific stretch of 17-1

9nucleotides.The two defined sequences are centered,respectively,at about 10 bp and at about 35 bp upstream of the site where RNA synthesis

starts.The sequences are thus called the –35 and

–10 regions,or elements.Position +1is the transcription start site.

Consensus sequence Although the vast majority of σ70 promoters

contain recognizable –35 and –10 regions,the sequences are not identical.

Comparison of many different sequences reflecting preferred –10 and –35 regions

Promoters with sequences closer to the consensus are generally “stronger” than those that match less well.

By the strength of a promoter,we mean how many transcripts it initiates in a given time.

BOX 12-1 Figure 1

Consensus sequence of the -35 and -10 region

Up-element An additional DNA element that binds RNA

polymerase is found in some strong promoters

Up-element can increases polymerase binding by providing an additional specific interaction between the enzyme and DNA

The magnificence is this : another class of σ70 –promoters lacks a –35region and instead gas a so called “extended-10” element,which compensates for the absence of –35 region.

UP-element is recognized by a carboxyl terminal domain of the -subunit (CTD), but not by factor

Fig 12-7 and subunits recruit RNA pol core enzyme to the promoter

Fig 12-5c bacterial promoter

Another class of 70 promoter lacks a –35 region and has an “extended –10 element” compensating for the absence of –35 region

2-2 The features of transcription in bacteria

1.Transition to the open complex involves structural changes in RNA pol and in the promoter DNA (melting , isomerization, the active center cleft)

2.Transcription is initiated by RNA pol without the need for a primer

3.RNA pol synthesizes several short RNAs before entering the elongation phase. (Abortive initiation)

4.The elongating pol is a processive machine that synthesizes and proofreads RNA.(pyrophosphorolytic editing & hydrolytic editing)

5.transcription is terminated by signals within the TNA sequence (Rho-independent Vs Rho-dependent, intrinsic terminators.)

Rho-independent terminator contains a short inverted repeat (~20 bp) and a stretch of ~8 A:T base pairs.

Fig 12-9Fig 12-9

Fig 12-11 the transcription terminator

Hexamer,

Open ring

RNA tread trough the “ring”

Topic 3 : transcription in eukaryotes

Transcription in bacteria Vs in eukaryotes

Eukaruotes have three different pol (I,II,III), whereas bacteria have only one.

Bacteria require only one additional initiation factor(σfactor ) , but several initiation factors are required for efficient and promoter-specific initiation in eukaryotes,which is called the general transcription factors(GTFs)

The factors needed for transcription in vivo

GTFs Polymerase Mediator complex DNA-binding regulatory proteins Chromatin-modifying enzymes

However ,in vitro, the general transcription factors are all that is required,together with pol II .

One reason for the difference is that the DNA template in vivo is packaged into nucleosomes and chromatin .This condition complicates binding to the promoter of pol and its associated factors.

Core promoter Core promoter refers to the

minimal set of sequence elements required for accurate transcription initiation by the pol II machinery.

A core promoter is typically about 40 nucleotides long, extending either upstream or downstream of the transcription start site.

TFIIB recognition element (BRE) The TATA element/box Initiator (Inr) The downstream promoter element (DPE)

Fig 12-12: Pol II core promoter

Fore elements in core promoter BRE : the TFIIB recognition element The TATA element Inr : the initiator DPE: the downstream promoterGenerally , a promoter includes only

two or three of these four elements .

Regulatory sequences Beyond the core promoter, there are other

sequence elements required for efficient transcription in vivo.These elements constitute the regulatory sequences.

They can be grouped into varous categories, reflecting their location, and the organism in question ,as much as their function

The regulatory sequences include Promoter proximal elements Upstream activator sequences (UASs) Enhancers A series of repressing elements called

silencers,boundary elements ,insulators .

All of them bind regulatory elements ,which help or hinder transcription .

Details of GTFs They can help pol bind to the

promoter and melt the DNA. Also help pol escape from the

promoter and embark on the elongation phase.

Pre-initiation complex = GTFs + promoter ,

can initiate the transcription .

Formation of pre-initiation complex

TFIID recognizes the TATA element TBP formed when TFIID binds to the

TATA element Another subunits on this complex

are called TAFs ,for TBP associated factors .

Other GTFs involved are TFIIA ,B, F,E, H

Something about TBP TBP binds to and distorts DNA using a

βsheet inserted into the minor groove ,while typically proteins recognize DNA using αhelices inserted into the major groove of DNA.

The reason for TBP’s unorthodox recognition mechanism is linked to the need for that protein to distort the local DNA structure.

TBP binds to and distorts DNA usiTBP binds to and distorts DNA using ang a sheet sheet inserted into inserted into the mithe minor groovenor groove

Unusual (P367 for the detailed mechanism)

The need for that protein to distort the local DNA structure

Th

e tra

nscrip

tion

in e

ukary

ote

s

TBP binds to and distorts DNA usiTBP binds to and distorts DNA using ang a sheet sheet inserted into inserted into the mithe minor groovenor groove

Unusual (P367 for the detailed mechanism)

The need for that protein to distort the local DNA structure

Th

e tra

nscrip

tion

in e

ukary

ote

s

The other GTFs also have specific roles in initiation

1.TAFs Two of them bind DNA elements at the

promoter;several of them have structural homology to

histone proteins :Another appears to regulate the binding of

TBP to DNA ,using an inhibitory 2.TFIIBThis protein ,a single polypeptide chain,enter

the pre-initiation complex after TBP

3.TFIIE It has two subunits ,associating with pol II an

d recruited to the promoter together with that enzyme.

4.TFIIE&TFIIH TFIIE,which ,like TFIIF, consists of two subuni

ts ,binds next and has roles in the recruitment and regulation of TFIIH,which controls the ATP-dependent transition of the pro-initiation complex to the open complex

The C-terminal Domain The contraction is CTD In the shape of tail Containing a series of the

heptapeptide sequence. Involved in the abortive initiation ,

promoter escape. Control later steps involving

processing of the RNA

Mediator complex Consists of many subunits (more than 20),

some conserved from yeast to human . There are 7 subunits of 20 ones showing

sequence homology between the two organisms.

Few of them have any identified function. Only one is essential for transcription of

essentially all pol II genes in vivo.

Fig 12-17 comparison of the yeast and human mediators

Fig 12-17 comparison of the yeast and human mediators

Fig 12-16 assembly of the pre-initiation complex in presence of mediator, nucleosome modifiers and remodelers, and transcriptional activators

RNA Pol II holoenzyme ? The dissociation arises the question th

at whether the RNA Pol II holoenzyme exists

The enzyme is a complex consisting of pol II,Mediator, and some of the GTFs

Sometimes ,the complex can be isolated from cells as a single one in the absence of DNA

Elongation factors

A new set of factors stimulate pol II elongation and RNA proofreading.

(1)CTD The phosphorylation of the CTD leads

to an exchange of initiation factors for those factors required for elongation and RNA processing.

Beside CDT,various proteins are thought to stimulate elongation by pol II :

The kinase P-TEFb :recruited to polymerase by transcriptional activators.

TAT-SF1 :recruited by P-TEFb TEIIS : does not affect initiation , but

stimulates elongation; contributes to proofreading by pol .

RNA processing Elongating pol is associated with a new set

of protein factors required for various types for RNA processing

Once transcribed, eukaryotic RNA has to be processed in various ways before being exported from the nucleus where it can be translated.

In fact ,elongation , termination of transcription,and RNA processing are interconnected-presumably to ensure their proper coordination

Fig 12-18 RNA processing enzymes are recruited by the tail of polymerase

The processing events include : Splicing :the most complicated Capping of the 5’ end of RNA :the firs

t RNA processing event ,involving the addition of a modified guanine base to the 5’ end of the RNA .

Polyadenylation of the 3’ end of the RNA :mediated by poly-A polymerase .

RNA processing 15’ end cappingRNA processing 15’ end capping The “cap”: a methylat

ed guanine joined to the RNA transcript by a 5’-5’ linkage

The linkage contains 3 phosphates

3 sequential enzymatic reactions

Occurs early

RNA processing 15’ end cappingRNA processing 15’ end capping The “cap”: a methylat

ed guanine joined to the RNA transcript by a 5’-5’ linkage

The linkage contains 3 phosphates

3 sequential enzymatic reactions

Occurs early

Splicing: joining the protein coding sequences Dephosphorylation of Ser5 within the CTD ta

il leads to dissociation of capping machinery Further phosphorylation of Ser2 recruits the

splicing machinery

1. CPSF (cleavage and polyadenylation specificity factor) & CstF (cleavage stimulation factor) bind to the poly-A signal, leading to the RNA cleavage 2. Poly-A polymerase (PAP) adds ~ 200 As at the 3’ end of the RNA, using ATP as a substrate

Fig 12-20 polyadenylation and termination

RNA Pol I&RNA Pol III RNA Pol I and III recognize distinct

promoters ,using distinct sets of transcription factors ,but still require TBP

Different from RNA Pol II, they transcribe distinct genes encoding specialized RNAs ,rather than proteins.

RNA Pol I Requred for the expression of only on

e gene ,that encoding the rRNA precursor .

The gene transcribed by RNA Pol I is expressed at a extremely high level.

Comprises of two parts : the core element & the UCE

Initiates with existence of SL1&UBF

Pol I promoter recognition

Fig 12-21 Pol I promoter region

Upstream control element

UBF binds to the upstream of UCE, bring SL1 to the downstream part of UCE. SL1 in turn recruits RNAP I to the core promoter for transcription

RNA Pol III Pol III promoters come in various for

ms,and the vast majority have the unusual feature of being located downstream of the transcription start site.

The factors required for transcription are called TFIIIB and TFIIIC ,and those plus TFIIIA for the 5S rRNA gene.

Fig 12-22 Pol III core promoter

TFIIIC binds to the promoter, recruiting TFIIIB, which in turn recruits RNAP III

Pol III promoter recognition1. Different forms, 2. locates downstream of the transcription site

SUMMARY RNA polymerase : crab claw structure &

function (mediated the transcription) Transcription Vs replication Transcription

cycle :initiation ,elongation, and termination

Transcription in bacteria (σfactor ) Transcription in eukaryotes (RNA pol I ,

II ,III ;GTFs , core promoters ;regulatory sequence )

Thank you for seeing ! Made by : 生物学基地班 罗晓 学号： 200431060057