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Welcome to Chapter 12 Mechanisms of transcription.

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1 Welcome to Chapter 12 Mechanisms of transcription

2 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

3 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.

4 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.

5 Question :why transcription is less accurate than replication? I think the difference makes good sense if we associate it with the results of the mistakes. 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 subsequent generations.

6 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.

7 Outline 1. RNA polymerase & Transcription cycle 2. The transcription cycle in bacteria 3.Transcription in eukaryotes

8 Topic 1: RNA polymerase & The transcription cycle

9 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.

10 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

11 Table 12-1: The subunits of RNA polymerases

12 “ Crab claw ” shape of RNAP (The shape of DNA pol is__) Active center cleft

13   ’’  RPB3 RPB11 RPB2 RPB1 RPB6 Fig 12-2 RNAP Comparison The same color indicate the homologous of the two enzymes prokaryotic eukaryotic 

14 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

15 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?

16 Transcription by RNA Pol proceeds in a series of steps Initiation Elongation Termination Let us go deep into the details

17 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 ’

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

19 Transcription Initiation Invoves 3 Defined Steps Form closed complex Form open complex Form stable ternaty complex

20 Fig 12-3-initiation Binding (closed complex) Promoter “ melting ” (open complex) Initial transcription

21 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.

22 Open complex DNA strands separate around the transcription site The transcription bubble forms

23 Stable ternary complex Enzyme escape the promoter once it gets further than the 10 bp Stable ternary complex contains enzyme,DNA and RNA Then the elongation phase comes

24 Process 2 : Elongation Begins when the enzyme has synthesized a short stretch of RNA (about 10 bp) The RNA pol undergoes further comformational 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

25 Fig 12-3-Elongation and termination Termination Elongation

26 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

27 Topic 2 :The Transcription Cycle In Bacteria

28 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

29 Fig 12-5a: bacterial promoter The distance is conserved  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

30 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

31 Fig 12-6: regions of  Region 4 recognizes -35 element Region 2 recognizes -10 element Region 3 recognizes the extended – 10 element

32 Figure 12-4,Holoenzyme=  factor + core enzyme In cell, RNA polymerase initiates transcription only at promoters. Who confers the polymerase binding specificity?

33 Take E.coli as a example In the case of E.coli,the predominant σfactor is calledσ 70 factor. Promoters recognized by σ 70 factor share the following characteristic structure:two conserved sequences,each of six nucleotides,are separated by a nonspecific stretch of 17-19nucleotides. 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.

34 Consensus sequence Although the vast majority of σ 70 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.

35 BOX 12-1 Figure 1 Consensus sequence of the -35 and -10 region

36 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 σ 70 – promoters lacks a – 35region and instead gas a so called “ extended-10 ” element,which compensates for the absence of – 35 region.

37 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

38 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

39 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)

40 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.)

41 Rho-independent terminator contains a short inverted repeat (~20 bp) and a stretch of ~8 A:T base pairs. Fig 12-9

42 Fig the  transcription terminator Hexamer, Open ring RNA tread trough the “ring”

43 Topic 3 : transcription in eukaryotes

44 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)

45 The factors needed for transcription in vivo GTFs Polymerase Mediator complex DNA-binding regulatory proteins Chromatin-modifying enzymes

46 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.

47 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.

48 TFIIB recognition element (BRE) The TATA element/box Initiator (Inr) The downstream promoter element (DPE) Fig 12-12: Pol II core promoter

49 Fore elements in core promoter BRE : the TFIIB recognition element The TATA element Inr : the initiator DPE: the downstream promoter Generally, a promoter includes only two or three of these four elements.

50 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

51 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.

52 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.

53 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

54 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.

55 TBP binds to and distorts DNA using a  sheet inserted into the minor groove Unusual (P367 for the detailed mechanism) The need for that protein to distort the local DNA structure The transcription in eukaryotes

56 TBP binds to and distorts DNA using a  sheet inserted into the minor groove Unusual (P367 for the detailed mechanism) The need for that protein to distort the local DNA structure The transcription in eukaryotes

57 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.TFIIB This protein,a single polypeptide chain,enter the pre-initiation complex after TBP

58 3.TFIIE It has two subunits,associating with pol II and recruited to the promoter together with that enzyme. 4.TFIIE&TFIIH TFIIE,which,like TFIIF, consists of two subunits,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

59 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

60 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.

61 Fig comparison of the yeast and human mediators

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

63 RNA Pol II holoenzyme ? The dissociation arises the question that 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

64 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.

65 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.

66 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

67 Fig RNA processing enzymes are recruited by the tail of polymerase

68 The processing events include : Splicing :the most complicated Capping of the 5 ’ end of RNA :the first 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.

69 RNA processing 1 5 ’ 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

70 RNA processing 1 5 ’ 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

71 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

72 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 polyadenylation and termination

73 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.

74 RNA Pol I Requred for the expression of only one 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

75 Pol I promoter recognition Fig 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

76 RNA Pol III Pol III promoters come in various forms,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.

77 Fig Pol III core promoter TFIIIC binds to the promoter, recruiting TFIIIB, which in turn recruits RNAP III Pol III promoter recognition 1. Different forms, 2. locates downstream of the transcription site

78 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 )

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


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