《分子生物学》课件CHAPTER12

1、1Molecular Biology of the Gene, 5/E - Watson et al. (2004)Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the GenomePart IV: RegulationPart V: MethodsThe revised central dogma2008RNA processingGene regulationCh 6: The structures of DNA and RNACh 8: The repli

2、cation of DNA基因组的保持基因组的表达3Ch 12: Mechanisms of transcription Ch 13: RNA splicingCh 14: TranslationCh 15: The genetic codePart III: Expression of the GenomeChapter 12: Mechanisms of TranscriptionRNA polymerase and the transcription cycleThe transcription cycle in bacteriaTranscription in eukaryotesTr

3、anscription by RNA polymerase I and IIIMolecular Biology CourseTranscription vs replication5Transcription is chemically and enzymatically very similar to DNA replication.6Some important differences:RNA is made of ribonucleotidesRNA polymerase catalyzes the reaction, which does not require a primer (

4、de novo synthesis)The RNA product does not remain base-paired to the template DNA strandLess accurate (error rate: 10-4)7Transcription selectively copies only certain parts of the genome and makes one to several hundred, or even thousand, copies of any given section of the genome. (Replication?) 8Fi

5、g 12-1 Transcription of DNA into RNATranscription bubble9Topic 1: RNA Polymerase and The Transcription CycleCHAPTER12: Mechanisms of TranscriptionSee the interactive animation10RNA polymerases come in different forms, but share many featuresRNA polymerases performs essentially the same reaction in a

6、ll cellsBacteria have only a single RNA polymerases while in eukaryotic cells there are three: RNA Pol I, II and IIIRNA polymerase and the transcription cycle11RNA Pol II is the focus of eukaryotic transcription, because it is the most studied polymerase, and is also responsible for transcribing mos

7、t genes-indeed, essentially all protein-encoding genesRNA Pol I transcribe the large ribosomal RNA precursor geneRNA Pol III transcribe tRNA gene, some small nuclear RNA genes and the 5S rRNA genes12Table 12-1: The subunits of RNA polymerases13The bacterial RNA polymeraseThe core enzyme alone synthe

8、sizes RNAaabbw14aabwRPB3RPB11RPB2RPB1RPB6Fig 12-2 RNAP ComparisonThe same color indicate the homologous of the two enzymesprokaryoticeukaryoticb15“Crab claw” shape of RNAP (The shape of DNA pol is_)Active center cleft16There are various channels allowing DNA, RNA and ribonucleotides (rNTPs) into and

9、 out of the enzymes active center cleft17Transcription by RNA polymerase proceeds in a series of stepsInitiationElongationTerminationRNA polymerase and the transcription cycle18InitiationPromoter: the DNA sequence that initially binds the RNA polymerase The structure of promoter-polymerase complex u

10、ndergoes structural changes to proceed transcription DNA at the transcription site unwinds and a “bubble” formsDirection of RNA synthesis occurs in a 5-3 direction (3-end growing)19Transcription initiation involves 3 defined stepsForming closed complexForming open complexPromoter escape: stable tern

11、ary complexRNA polymerase and the transcription cycle20 The initial binding of polymerase to a promoter DNA remains double strandedThe enzyme is bound to one face of the helixClosed complex21Open complexthe DNA strand separate over a distance of 14 bp (-11 to +3 ) around the start site (+1 site)tran

12、scription bubble forms 22Stable ternary complexThe enzyme escapes from the promoterThe transition to the elongation phase Stable ternary complex =DNA +RNA + enzyme23Fig 12-3-initiationBinding (closed complex)Promoter “melting” (open complex)Initial transcription24ElongationOnce the RNA polymerase ha

13、s synthesized a short stretch of RNA ( 10 nt), transcription shifts into the elongation phase.This transition requires further conformational change in polymerase that leads it to grip the template more firmly.Functions: synthesis RNA, unwinds the DNA in front, re-anneals it behind, dissociates the

14、growing RNA chain 25TerminationAfter the polymerase transcribes the length of the gene (or genes), it will stop and release the RNA transcript.In some cells, termination occurs at the specific and well-defined DNA sequences called terminators. Some cells lack such termination sequences.26Fig 12-3-El

15、ongation and terminationTerminationElongation27Topic 2The transcription cycle in bacteriaCHAPTER12: Mechanisms of Transcription282-1 Bacterial promoters vary in strength and sequences, but have certain defining features The transcription cycle in bacteria29Figure 12-4,Holoenzyme= factor + core enzym

16、e In cell, RNA polymerase initiates transcription only at promoters. Who confers the polymerase binding specificity?30 The predominant s factor in E. coli is s70. Promoter recognized by s70 contains two conserved sequences (-35 and 10 regions/elements) separated by a non-specific stretch of 17-19 nt

17、. Position +1 is the transcription start site.Promoters recognized by E. coli s factor31Fig 12-5a: bacterial promoterThe distance is conserveds70 promoters contain recognizable 35 and 10 regions, but the sequences are not identical. Comparison of many different promoters derives the consensus sequen

18、ces reflecting preferred 10 and 35 regions32BOX 12-1 Figure 1Consensus sequence of the -35 and -10 region33Promoters with sequences closer to the consensus are generally “stronger” than those match less well. (What does “stronger” mean?)The strength of the promoter describes how many transcripts it

19、initiates in a given time.34Fig 12-5b bacterial promoterConfers additional specificityUP-element is an additional DNA elements that increases polymerase binding by providing the additional interaction site for RNA polymerase35Fig 12-5c bacterial promoterAnother class of s70 promoter lacks a 35 regio

20、n and has an “extended 10 element” compensating for the absence of 35 region362-2 The s factor mediates binding of polymerase to the promoter The transcription cycle in bacteriaThe s70 factor comprises four regions called s region 1 to s region 4.37Fig 12-6: regions of sRegion 4 recognizes -35 eleme

21、nt Region 2 recognizes -10 elementRegion 3 recognizes the extended 10 element38Binding of 35 Two helices within region 4 form a common DNA-binding motif, called a helix-turn-helix motifFig 5-20* Helix-turn-helix DNA-binding motifOne helix inserts into the DNA major groove interacting with the bases

22、at the 35 region. The other helix lies across the top of the groove, contacting the DNA backbone39Interaction with 10 is more elaborate (精细) and less understood The -10 region is within DNA melting regionThe a helix recognizing 10 can interacts with bases on the non-template strand to stabilize the

23、melted DNA. 40UP-element is recognized by a carboxyl terminal domain of the a-subunit (aCTD), but not by s factorFig 12-7 s and a subunits recruit RNA pol core enzyme to the promoter412-3 Transition to the open complex involves structural changes in RNA polymerase and in the promoter DNAThis transit

24、ion is called Isomerization (异构化) The transcription cycle in bacteria42 For s70 containing RNA polymerase, isomerization is a spontaneous conformational change in the DNA-enzyme complex to a more energetically favorable form. (No extra energy requirement) 43the opening of the DNA double helix, calle

25、d “melting”, at positions -11 and +3.Change of the promoter DNA44The striking structural change in the polymerase1. the b and b pincers down tightly on the downstream DNA2. A major shift occurs in the N-terminal region of s (region 1.1) shifts. In the closed complex, s region 1.1 is in the active ce

26、nter; in the open complex, the region 1.1 shift to the outside of the center, allowing DNA access to the cleft45NTP uptake channel is in the backFig 12-8 channels into and out of the open complexDNA entering channel46Transcription is initiated by RNA polymerase without the need for a primerInitiatio

27、n requires:The initiating NTP (usually an A) is placed in the active siteThe initiating ATP is held tightly in the correct orientation by extensive interactions with the holoenzyme The transcription cycle in bacteria47RNA polymerase synthesizes several short RNAs before entering the elongation phase

28、Abortive initiation: the enzyme synthesizes and releases short RNA molecules less than 10 nt. The transcription cycle in bacteria48Structural barrier for the abortive initiationThe 3.2 region of s factor lies in the middle of the RNA exit channel in the open complex.Ejection of this region from the

29、channel (1) is necessary for further RNA elongation, (2) takes the enzyme several attempts49NTP uptake channel is in the backFig 12-8 channels into and out of the open complexDNA entering channel50The elongating polymerase is a processive machine that synthesizes and proofreads RNA The transcription

30、 cycle in bacteria51DNA enters the polymerase between the pincersStrand separation in the catalytic cleftNTP additionRNA product spooling out (Only 8-9 nts of the growing RNA remain base-paired with the DNA template at any given time) DNA strand annealing in behindSynthesizing by RNA polymerase52Pyr

31、ohosphorolytic （焦磷酸键解）editing: the enzyme catalyzes the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi.Hydrolytic （水解）editing: the enzyme backtracks by one or more nucleotides and removes the error-containing sequence. This is stimulated by Gre factor, a elongation stimu

32、lation factor.Proofreading by RNA polymerase53Transcription is terminated by signals within the RNA sequenceTerminators: the sequences that trigger the elongation polymerase to dissociate from the DNARho-dependent (requires Rho protein)Rho-independent, also called intrinsic (内在) terminator The trans

33、cription cycle in bacteria54Rho-independent terminator contains a short inverted repeat (20 bp) and a stretch of 8 A:T base pairs. Fig 12-1255Weakest base pairing: A:U make the dissociation easierFig 12-13 transcription termination56Rho (r) -dependent terminatorsHave less well-characterized RNA elem

34、ents, and requires Rho protein for terminationRho is a ring-shaped single-stranded RNA binding protein, like SSBRho binding can wrest (夺取) the RNA from the polymerase-template complex using the energy from ATP hydrolysisRho binds to rut (r utilization) RNA sites Rho does not bind the translating RNA

35、57Fig 12-11 the r transcription terminator Hexamer,Open ringRNA tread trough the “ring”58Topic 3:transcription in eukaryotesCHAPTER12: Mechanisms of Transcription59A comparison with bacterial transcription60Comparison of eukaryotic and prokaryotic RNA polymerasesEukaryotes: Three polymerase transcri

36、bes different class of genes: Pol I-large rRNA genes; Pol II-mRNA genes; Pol III- tRNA, 5S rRNA and small nuclear RNA genes (U6) Prokaryotes: one polymerase transcribes all genes61Comparison of eukaryotic and prokaryotic promoter recognitionEukaryotes: general transcription factors (GTFs) (sufficien

37、t for initiate the transcription by RNA Pol in vitro but not in vivo). TFI factors for RNAP I, TFII factors for RNAP II and TFIII factors for RNAP III Prokaryotes: s factors62In addition to the RNAP and GTFs, in vivo transcription also requiresMediator complexDNA-binding regulatory proteinschromatin

38、-modifying enzymesWhy?63Cis-acting elements: core promoter & regulatory sequencesFormation of the pre-initiation complexPromoter escapeThe function of each GTF (in vitro)Additional proteins for in vivo transcription. Transcription initiation for RNA Pol II64RNA polymerase II core promoters are made

39、up of combinations of 4 different sequence elementsEukaryotic core promoter (40-60 nt): the minimal set of sequence elements required for accurate transcription initiation by the Pol II machinery in vitro The transcription in eukaryotes65TFIIB recognition element (BRE)The TATA element/boxInitiator (

40、Inr)The downstream promoter elements (DPE, DCE, MTE)Fig 12-12: Pol II core promoter66The sequence elements other than the core promoter that are required to regulate the transcription efficiency.Those increasing transcription:Promoter proximal elementsUpstream activator sequences (UASs)EnhancersThos

41、e repressing elements: silencers, boundary elements, insulators (绝缘体)Regulatory sequences67RNA Pol II forms a pre-initiation complex with GTFs at the promoterThe involved GTFIIs (general transcription factor for Pol II)TFIID=TBP (TATA box binding protein) + TAFs (TBP association factors) TFIIA, B, F

42、, E, H The transcription in eukaryotes68TBP in TFIID binds to the TATA boxTFIIA and TFIIB are recruited with TFIIB binding to the BRERNA Pol II-TFIIF complex is then recruitedTFIIE and TFIIH then bind upstream of Pol II to form the pre-initiation complex Promoter melting using energy from ATP hydrol

43、ysis by TFIIH )Promoter escapes after the phosphorylation of the CTD tail.Promoter escape requires phosphorylation of the polymerase “tail”Stimulated by phosphorylation of the CTD (C-terminal domain) tail of the RNAP IICTD contains the heptapeptide repeat Tyr-Ser-Pro-Thr-Ser-Pro-SerPhosphorylation o

44、f the CTD “tail” is conducted by a number of specific kinases including a subunit of TFIIH The transcription in eukaryotesKinase vs Phosphotase70TBP binds to and distorts DNA using a b sheet inserted into the minor grooveUnusual (P367 for the detailed mechanism)The need for that protein to distort t

45、he local DNA structure The transcription in eukaryotes71A:T base pairs (TATA box) are favored because they are more readily distorted to allow initial opening of the minor groove72The other GTFs also have specific roles in initiation10 TAFs in TFIID: (1) two of them bind DNA elements at the promoter

46、 (Inr and DPE); (2) several are histone-like TAFs and might bind to DNA similar to that histone does; (3) one regulates the binding of TBP to DNA. The transcription in eukaryotes73TFIIB: (1) a single polypeptide chain, (2) base specific interaction with the major groove of BRE, (3)Binding to TBP-TAT

47、A complex asymmetrically, which accounts for the unidirectional transcription. (4) Also contacts RNA Pol II-a bridge.Fig 12-17 TFIIB-TBP-promoter complex74TFIIF: (1) a two subunit factor, (2) Associates and recruits RNA Pol II to the promoter, and this binding stabilizes the DNA-TBP-TFIIB complex, w

48、hich is required for the followed factor bindingTFIIE: recruits and regulates TFIIHTFIIH: (1) controls the ATP-dependent transition of the pre-initiation complex to the open complex, (2) contains 9 subunits and is the largest GTF; two function as ATPase and one is protein kinase. (3) important for p

49、romoter melting and escape. (4) ATPase also functions in nucleotide mismatch repair, called transcription-coupled repair. 75in vivo, transcription initiation requires additional proteinsThe mediator complexTranscriptional regulatory proteinsNucleosome-modifying enzymesTo counter the real situation t

50、hat the DNA template in vivo is packed into nucleosome and chromatin The transcription in eukaryotes76Fig 12-18 assembly of the pre-initiation complex in presence of mediator, nucleosome modifiers and remodelers, and transcriptional activators77Mediator consists of many subunits, some conserved from

51、 yeast to human.More than 20 subunits7 subunits show significant sequence homology between yeast and humanOnly subunit Srb4 is essential for transcription of essentially all Pol II genes in vivo.Organized in modules (模块) The transcription in eukaryotes78Fig 12-19 comparison of the yeast and human me

52、diators79Transcription elongation 80Elongating polymerase must deal with histons in its path. The transcription in eukaryotesFigure 12-2281A new set of factors stimulate Pol II elongation and RNA proofreading The transcription in eukaryotesA brief comparison with bacterial polymerase elongation82Tra

53、nsition from the initiation to elongation involves the Pol II enzyme shedding (摆脱) most of its initiation factors (GTF and mediators) and recruiting other factors: (1) Elongation factors: factors that stimulate elongation, such as TFIIS and hSPT5.(2) RNA processing (RNA 加工) factors Phosphoylation of

54、 the CTD leads to an exchange of initiation factors for those factors required for elongation and RNA processing.83Various proteins are thought to stimulate elongation by Pol II P-TEFb (a kinase stimulating elongation in 3 separate ways): 1. Phosphorylates the serine residue at position 2 of the CTD

55、 repeats.2. Activates hSPT5.3. Recruits TAT-SF1.TFIIS: (1) Stimulates the overall rate of elongation by resolving the polymerase pausing. (2) Proofreading (Fig. 12-21)84Fig 12-21 TFIIS and GreB act in analogous way85Elongation polymerase is associated with a new set of protein factors required for v

56、arious types of RNA processing.RNA processing: Capping of the 5 end of the RNASplicing to remove noncoding introns (Chapter 13)Polyadenylation (多聚腺苷化) of the 3 end The transcription in eukaryotes86Function of 5cap and 3 poly(A) tailProtection of mRNA from degradation by exonucleases (5-3 or 3-5).Inc

57、rease translational efficiency.5 cap facilitates mRNA transport to cytoplasm.Facilitate the splicing of first intron by 5 cap and the last intron by poly(A) tail. AAAAAA87Fig 12-20 RNA processing enzymes are recruited by the tail of polymeraseDephosphorylation of Ser5 within the CTD tail leads to di

58、ssociation of capping machinery Further phosphorylation of Ser2 recruits the splicing machinery 88Evidence: this is an overlap in proteins involved in elongation and those for RNA processing.The elongation factor hSPT5 also recruits and stimulates the 5 capping enzyme.The elongation factor TAT-SF1 r

59、ecruits 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 events89RNA processing 15 end cappingThe “cap”: a methylated guanine joined to the RNA transcript by a 5-5 linkageThe linkage cont

60、ains 3 phosphates3 sequential enzymatic reactionsOccurs early 90Linked with the termination of transcriptionThe CTD tail is involved in recruiting the polyadenylation enzymes.The transcribed poly-A signal triggers the reactions:Cleavage of the messageAddition of poly-ATermination of transcription RN