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Chapter 12: Mechanisms Chapter 12: Mechanisms of Transcriptionof Transcription

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RNA polymerase and transcription cycle

The transcription cycle in bacteria

Transcription in eukaryotes

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Mechanistic features of transcription is very similar to DNA replication but there are some important differences:

RNA polymerase does not need a primer.The RNA product does not remain base-

paired to the template DNA strand.Transcription, though very accurate , is

less accurate than replication.

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RNA Polymerase and The Transcription Cycle

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RNA polymerases come in different forms, RNA polymerases come in different forms, but share many featuresbut share many features

Transcription by RNA polymerase proceeds Transcription by RNA polymerase proceeds in a series of stepsin a series of steps

Transcription initiation involves 3 defined Transcription initiation involves 3 defined stepssteps

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1.RNA polymerases performs

essentially the same reaction in all cells

Table 12-1: The subunits of RNA polymerases

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2.Bacteria have only a single RNA polymerases while in eukaryotic cells there are three: RNA Pol I, II and III

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’

RPB3

RPB11

RPB2

RPB1

RPB6

prokaryotic

eukaryotic

Figure 12-2 Comparison of the crystal structures of prokaryotic and eukaryotic RNA polymerases

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3.To transcribe a gene , RNA 3.To transcribe a gene , RNA polymerase proceeds through a polymerase proceeds through a series of well-defined steps which are series of well-defined steps which are grouped into three phases:grouped into three phases:

Initiation Elongation Termination

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initiation

Elongation

Termination

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Initiation

Promoter.

promoter-polymerase complex undergoes structural changes required for initiation to proceed.

DNA at the transcription site unwinds and a “bubble” forms

Direction of RNA synthesis occurs in a 5’-3’ direction (3’-end growing)

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Binding (closed complex)

Promoter “melting” (open complex)

Initial transcription

Figure 12-3-initiation

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Elongation1. Once the RNA polymerase has

synthesized a short stretch of RNA ,it shifts into the elongation phase.

2. During elongation , the enzyme performs an impressive range of tasks in addition to the catalysis of RNA synthesis, it unwinds the DNA in front and re-anneals it behind, it dissociates the growing RNA chain from the template as it moves along ,and it performs proofreading functions.

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Termination

Once the polymerase has transcribed the length of the gene (or genes), it must stop and release the RNA product. This step is called Termination

In some cells there are specific, well-characterized, sequences that trigger termination; but in others it is less clear.

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Termination

Elongation

Figure 12-3-Elongation and termination

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Forming closed complex

Forming open complex

Promoter escape

4.The first phase in the transcription cycle-initiation-can itself be broken down into a series of defined steps:

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The initial binding of polymerase to a promoter

DNA remains double strandedThe enzyme is bound to one

face of the helix

Closed complex

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The transcription cycle in bacteria

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1.Bacterial promoters vary in 1.Bacterial promoters vary in strength and sequences, but have strength and sequences, but have certain defining featurescertain defining features

In cells polymerase initiates transcription only at promoters. It is the addition of an initiation factor called s that converts core enzyme into the form that initiates only at promoters. That form of the enzyme is called the RNA polymerase holoenzyme (Figure 12-4)

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,

Figure 12-4 RNA polymerase holoenzyme T.aquaticus.

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Two conserved sequences each of six nucleotides, are separated by a non-specific stretch of 17-19 nucleotides (Figure 12-5) .

The two defined sequences are centered, respectively, at about 10 base pairs and at about 35 base pairs upstream of the site where RNA synthesis starts.

Position +1 is the transcription start site. The sequences are thus called -35(minus 35) and –10(minus 10) regions, or elements.

2.In the case of E. coli ,the predominant s factor is called s70. They share the following characteristic structure:

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Figure 12-5: Features of bacterial promoters

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An additional DNA element that binds RNA polymerase is found in some strong promoters, for example those directing expression of the ribosomal RNA (rRNA) genes This is called an UP-element (Figure 12-5b)and increases polymerase binding by providing an additional specific interaction between the enzyme and the DNA.

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Another class of 70 promoter lacks a –35 region and instead has an “extended –10 element” compensating for the absence of –35 region. (Figure 12-5c)

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3.The 3.The factor mediates factor mediates binding of polymerase to the binding of polymerase to the promoterpromoter

The 70 factor comprises four regions called region 1 to region 4.

The regions that recognize the -10 and -35 elements of the promoter are region 2 and 4, respectively.

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Figure 12-6: regions of s

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4.Two helices within region 4 form a common DNA-binding motif called a helix-turn-helix:

One of these helices inserts into the major groove and interacts with bases in the -35 region;

The other lies across the top of the groove, making contacts with the DNA backbone.

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The -10 region is also recognized by an helix.

The region of that interacts with the -10 region is doing more than simply binding DNA.

The helix involved in recognition of the -10 region contains several essential aromatic amino acids that can interacts with bases on the non-template strand to stabilize the melted DNA.

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UP-element is

recognized by a carboxyl terminal

domain of the -subunit

(CTD), but not by factor

Figure 12-7 and subunits recruit RNA polymerase core enzyme to the promoter

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5.Transition to the open complex 5.Transition to the open complex involves structural changes in RNA involves structural changes in RNA polymerase and in the promoter DNApolymerase and in the promoter DNA

The initial binding of RNA polymerase to the promoter DNA in the closed complex leaves the DNA in double –stranded form .

In the case of the bacterial enzyme bearing 70,this transition, often called isomerizationdoes not require energy derived from ATP hydrolysis.

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6.The structure of holoenzyme in more detail A channel runs between the pincers of the

claw-shaped enzyme. The active site of the enzyme, which is

made up of regions from both the and’ subunits, is found at the base of the pincers within the “active center cleft.”

There are five channels into the enzyme, as shown in the picture of the open complex in Figure 12-8.

32Figure 12-8 channels into and out of the open

complex

The NTP-uptake channel allows

ribonucleotides to enter the active

center . The RNA-exit channel

allows the growing RNA chain to leave the enzyme as it is

synthesized during elongation.

The remaining three channels

allow DNA entry and exit from the

enzyme, as follows.

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7.Two striking structural change in the polymerase

First, the pincers at the front of the enzyme clamp down tightly on the downstream;

Second, there is a major shift in the N-terminal region of (region 1.1) shifts. In the closed complex, region 1.1 is in the active center; in the open complex, the region 1.1 shift some 50 Å to the outside of the center, allowing DNA access to the cleft

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8.Transcription is initiated by RNA 8.Transcription is initiated by RNA polymerase without the need for a polymerase without the need for a primerprimer

RNA polymerase can initiate a new RNA chain on a DNA template and thus does not need a primer.

The enzyme has to make specific interactions with the initiating ribonucleotide, holding it rigidly in the correct orientation to allow chemical attack on the incoming NTP.

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9.RNA polymerase synthesizes 9.RNA polymerase synthesizes several short RNAs before entering several short RNAs before entering the elongation phasethe elongation phase

Abortive initiation: In this phase, the enzyme synthesizes short RNA molecules of less than ten nucleotides in length. Instead of being elongated further, these transcripts are released from the polymerase, and the enzyme, without disassociating from the template, begins RNA synthesis again.

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It is not clear why RNA polymerase undergoes this period of abortive initiation. but once again a region of the s factor appears to be involved, acting as a molecular mimic.

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10.The elongating polymerase is 10.The elongating polymerase is a processive machine that a processive machine that synthesizes and proofreads RNAsynthesizes and proofreads RNA

At the opening of the catalytic cleft, the strands separate to follow different paths through the enzyme before exiting via their respective channels and reforming a double helix behind the elongating polymerase.

Ribonucleotides enter the active site through their defined channel and are DNA strand.

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Proofreading by RNA polymerase

Pyrohosphorolytic editing （焦磷酸化编辑 ): the enzyme uses its active site, to catalyze the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi. Then it incorporate another ribonucleotide in its place in the growing RNA chain.Hydrolytic （水解） editing: the polymerase backtracks by one or more nucleotides and cleaves the RNA product, removing the error-containing sequence.

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11.Transcription is terminated by 11.Transcription is terminated by signals within the RNA sequencesignals within the RNA sequence

Sequences called terminators trigger the elongation polymerase to dissociate from the DNA and release the RNA chain it has made.

There are two types of terminators in bacteria:Rho-dependent Rho-independent (intrinsic terminator)

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Rho-independent terminator

contains a short inverted repeat followed by a

stretch of about 8 A:T base

pairs. Figure 12-9 Sequence of a rho-independent terminator

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Figure 12-10 transcription termination

Shown is a model for how

the rho-independent

terminator might work.

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12.Once attached to the transcript, Rho uses the energy derived from ATP hydrolysis to wrest the RNA from the template and from polymerase.

Rho is directed to a particular RNA molecule:

There is some specificity in the sites it binds. These sites are rich in C residues.

Rho fails to bind any transcript that is being translated.

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Figure 12-11 the transcription terminator

The crystal structure of

the rho termination

factor is shown in a top down

view.

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transcription in eukaryotes

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1.RNA polymerase II core 1.RNA polymerase II core promoters promoters are made up of are made up of combinations of 4 different combinations of 4 different sequence elementssequence elementsFigure 12-2 shows the location, relative to

the transcription start site, of four elements found in Pol core promoters:Ⅱ

TFIIB recognition element (BRE)TATA element (or box) Initiator (Inr)Downstream promoter element (DPE)

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Figure 12-12: Pol II core promoter

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Pre-initiation complex: The complete set of general transcription factors and polymerase, bound together at the promoter and poised for initiation

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Some TAFs help bind the DNA at certain promoters, and others control the DNA-binding activity of TBP.

Upon binding DNA, TBP extensively distorts the TATA sequence.

TFIIA, TFIIB, TFIIF together with polymerase, and then TFIIE and TFIIH,

which bind upstream of Pol .Ⅱ

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FIGURE 12-13 Transcription initiation by

RNA polymerase Ⅱ

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2.TBP binds to and distorts DNA using 2.TBP binds to and distorts DNA using a b sheet inserted into the minor a b sheet inserted into the minor groovegroove

When it binds DNA, TBF causes the minor groove to be widened to an almost flat conformation;

It also bends the DNA by an angle of approximately 80°

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3.The other GTFs also have 3.The other GTFs also have specific roles in initiationspecific roles in initiation

TAFs: TBP is associated with about ten TAFs. Two of the TAFs bind DNA elements at the promoter. Another TAF appears to regulate the binding of TBP to DNA.

52Figure 12-15 TFIIB-TBP-promoter complex

TFIIB: This protein, a single polypeptide chain, enters the preinitiation complex

after TBP

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TFIIF: This two-subunit factor associates with that enzyme. Binding of Pol -TFIIF stabilizes the DNA-TBP-ⅡTFIIB complex and is required before TFIIE and TFIIH are recruited to the pre-initiation complex.

TFIIE and TFIIH: TFIIE, which, like TFIIF, consists if two subunits, binds next, and has roles in the recruitment and regulation of TFIIH.

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4.in vivo, transcription initiation 4.in vivo, transcription initiation requires additional proteins, requires additional proteins, including the mediator complexincluding the mediator complex The DNA template in vivo is packaged into

nucleosomes and chromatin. This condition complicates binding to the promoter of polymerase and its associated factors.

Different activators are believed to interact with different Mediator aids initiation by regulating the CTD kinase in TFIIH

The need for nucleosome modifiers and remodellers also differs at different promoters or even at the same promoter under different circumstances.

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5.Mediator consists of many 5.Mediator consists of many subunits, some conserved from subunits, some conserved from yeast to humanyeast to human

The yeast and human Mediator each include more than 20 subunits, of which 7show significant sequence homology between the two organisms.

Figure 12-17 comparison of the yeast and human

mediators

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A new set of factors stimulate Pol II A new set of factors stimulate Pol II elongation and RNA proofreadingelongation and RNA proofreading

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

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6.Some elongation factors

P-TEFb: phosphorylates CTDActivates hSPT5Activates TAT-SF1

TFIIS:Stimulates the overall rate of elongation by

resolving the polymerase pausingProofreading

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7.Elongation polymerase is 7.Elongation polymerase is associated with a new set of associated with a new set of protein factors required for protein factors required for various types of RNA various types of RNA processingprocessing

RNA processing: Capping of the 5’ end of the RNASplicing of the introns (most complicated)Poly adenylation (多聚腺苷化 ) of the 3’ end

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Evidence: this is an overlap in proteins involving in those events The elongation factor hSPT5 also

recruits and stimulates the 5’ capping enzyme

The elongation factor TAT-SF1 recruits components for splicing

Elongation, termination of transcription, and RNA processing are interconnected/ coupled ( 偶联的 ) to ensure the coordination ( 协同性 ) of these events

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RNA processing 15’ end capping

RNA processing 15’ end capping

The “cap”: a methylated guanine joined to the RNA transcript by a 5’-5’ linkage

The linkage contains 3 phosphates

3 sequential enzymatic reactions

Occurs early

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Splicing: joining the protein coding sequences

Dephosphorylation of Ser5 within the CTD tail leads to dissociation of capping machinery

Further phosphorylation of Ser2 recruits the splicing machinery

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3’ end polyadenylation

Linked with the termination of transcription

The CTD tail is involved in recruiting the polyadenylation enzymes

The transcribed poly-A signal triggers the reactions

1. Cleavage of the message

2. Addition of poly-A

3. Termination of transcription

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Figure 12-20 polyadenylation and

termination

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8.Models to explain the link between polyadenylation and termination

First model: The transfer of the 3’ processing enzymes to RNAP II induces conformational change—RNAP II processivity reduces—spontaneous termination

Second model: absence of a 5’cap on the second RNA molecule—recognized by the RNAP II as improper—terminate transcription

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9.RNA Pol I & III recognize distinct 9.RNA Pol I & III recognize distinct promoters , using distinct sets of promoters , using distinct sets of transcription factors, but still transcription factors, but still require TBPrequire TBP

Pol I is required for the expression of only one gene, that encoding the rRNA precursor.

Pol III promoters come in various forms, and the vast majority have the unusual feature of being bocated downstream of the transcription start site.

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Pol I promoter recognition

Fig 12-21 Pol I promoter region

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Figure 12-22 Pol III core promoter

Show here is the promoter for a yeast tRNA gene.The order of events leading to transcription initiation is described in the text.

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