分子生物学教学课件：CHAPTER 12 Mechanisms of transcription

1、1Chapter 12: 2Molecular Biology of the Gene, - Watson et al. -Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the GenomePart IV: RegulationPart V: Methods345Francis Crick Cricks Most Important Contributions: 1) Established basic genetics codes;2) Predicted t

2、he mechanism for protein synthesis; 3) Which then led to the DNA/ RNA dictionary; 4) He also discovered double helix.1958年，年，Crick 阐明了中心法则阐明了中心法则6Mechanistic features of transcription is very similar to DNA replication but there are some important differences: RNA is made of ribonucleotides. RNA pol

3、ymerase catalyzes the reaction and 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.7Transcription selectively copies only certain parts of the genome and makes one to several hundr

4、ed, or even thousand, copies of any given section of the genome. (Replication?) 8Fig 12-1 Transcription of DNA into RNATranscription bubble910111213a aa ab bw wRPB3RPB11RPB2RPB1RPB6Fig 12-2 RNAP ComparisonThe same color indicate the homologous of the two karyoticeukaryoticb b14Active cent

5、er cleftThere are various channels allowing DNA, RNA and ribonucleotides (rNTPs) into and out of the enzymes active center cleft151617Binding (closed complex)Promoter “melting” (open complex)Initial transcriptionFigure 12-3 Initiation18ElongationnOnce the RNA polymerase has synthesized a short stret

6、ch of RNA ( 10 nt), transcription shifts into the elongation phase.nThis transition requires further conformational change in polymerase that leads it to grip the template more firmly.nFunctions: synthesis RNA, unwinds the DNA in front, re-anneals it behind, dissociates the growing RNA chain, proofr

7、eading. 1920Figure 12-3-Elongation and terminationTerminationElongation2122Closed complex232425InitiationElongationTermination2.The transcription cycle in bacteria1.Bacterial promoters2. 3. Closed /Open Complex4. Transcription Initiatin1. RNA synthesis 2.proofreading1. Rho-independent2.Rho-dependent

8、27In cells polymerase initiates transcription only at promoters. It is the addition of an initiation factor called s s that converts core enzyme into the form that initiates only at promoters. That form of the enzyme is called the RNA polymerase holoenzyme. 28In cell, RNA polymerase initiates transc

9、ription only at promoters. Who confers the polymerase binding specificity?s亚基 核心酶29Promoters recognized by E. coli s s factor301. 1. s s70 promoters contain recognizable 35 and 10 regions, but the sequences are not identical. 2.Comparison of many different promoters derives the consensus sequences r

10、eflecting preferred 10 and 35 regions.31Consensus sequence of the -35 and -10 region323.Promoters with sequences closer to the consensus are generally “stronger” than those match less well. (What does “stronger” mean?)4.The strength of the promoter describes how many transcripts it initiates in a gi

11、ven time.33 UP-element is an additional DNA elements that increases polymerase binding by providing the additional interaction site for RNA polymerase.34 Another class of s s70 promoter lacks a 35 region and has an “extended 10 element” compensating for the absence of 35 region.35nThe s s70 factor c

12、omprises four regions called s s region 1 to s s region 4.36Fig 12-6: Regions of s sRegion 4 recognizes -35 element Region 2 recognizes -10 elementRegion 3 recognizes the extended 10 element37One helix inserts into the DNA major groove interacting with the bases at the 35 region. The other helix lie

13、s across the top of the groove, contacting the DNA backboneFig 5-20* Helix-turn-helix DNA-binding motifOne helix inserts into the DNA major groove interacting with the bases at the 35 region. The other helix lies across the top of the groove, contacting the DNA backbone.3839Fig 12-7 s s and a a subu

14、nits recruit RNA pol core enzyme to the promoter4041n For s 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) 42nthe opening of the DNA double helix, called “melting”, at

15、 positions -11 and +3.Change of the promoter DNA4344NTP uptake channel is in the backDNA entering channel4546P391 Fig.12-947NTP uptake channel is in the backDNA entering channelInitiationElongationTermination2.The transcription cycle in bacteria1.Bacterial promoters2. 3. Closed /Open Complex4. Trans

16、cription Initiatin1.RNA synthesis 2.proofreading1. Rho-independent2.Rho-dependent4950DNA 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)

17、DNA strand annealing in behindSynthesizing by RNA polymerase51Proofreading by RNA polymerase52Fig 12-12RNA聚合酶延伸复合体内部的模板和转录物：a)未移位的聚合酶（0）与模板链9个碱基配对；b)（+1)显示聚合酶向前移动了一个碱基；c)结合NTP的前移聚合酶；d)如当水解编辑时，聚合酶回移。InitiationElongationTermination2.The transcription cycle in bacteria1.Bacterial promoters2. 3. Closed

18、/Open Complex4. Transcription Initiatin1. RNA synthesis 2.proofreading1. Rho-independent2.Rho-dependent5455Fig 12-1256Weakest base pairing: A:U make the dissociation easierFigure 12-13 transcription termination5758A hexamer of Rho protein; the six monomers form in an open ring.transc.swf59606162Init

19、iationElongationTermination3.The transcription cycle in eukaryotes1. Core promoters2.Proteins neededRNA Polymerase I and IIIRNA Polymerase 1. RNA elongation factors (proofreading)6465Fig 12-14: Pol II core promoter66Regulatory sequences671.Binds first.2.Contains and .3.: Recognition of the and possi

20、bly Inr sequence, form a platform for TFB binding. TFIID binds to the TATA box are recruited with TFIID binding to the BRE complex is then recruited then bind of Pol II to form the pre-initiation complex using energy from ATP hydrolysis by TFIIH after the phosphorylation of the CTD tail69707172nA:T

21、base pairs (TATA box) are favored because they are more readily distorted to allow initial opening of the minor groove.7374nTFIIB: (1) a single polypeptide chain, (2) asymmetric binding to TBP and the promoter DNA (BRE), (3) bridging TBP and the polymerase, (4) the N-terminal inserting in the RNA ex

22、it channel resembles the s s3.2 .3.2 .Fig 12-13 TFIIB-TBP-promoter complex757677787980Eukaryotic RNA Pol II holoenzyme is a putative preformed complex:Pol II + mediator + some of GTFs Prokaryotic RNA Polymerase holoenzyme:core polymerase + s s factorInitiationElongationTermination3.The transcription

23、 cycle in eukaryotes1. Core promoters2.Proteins neededRNA Polymerase I and IIIRNA Polymerase 2.RNA elongation factors (proofreading)8283nTransition from the initiation to elongation involves the Pol II enzyme shedding (摆脱摆脱) most of its initiation factors (GTF and mediators) and recruiting other fac

24、tors: (1) Elongation factors: factors that stimulate elongation, such as TFIIS and hSPT5.(2) RNA processing (RNA 加工加工) factors nRecruited to the C-terminal tail of the CTD of RNAP II to phosphorylate the tail for elongation stimulation, proofreading, and RNA processing like splicing and polyadenylat

25、ion.848586878889Evidence: this is an overlap in proteins involving in those events nThe elongation factor hSPT5 also recruits and stimulates the 5 capping enzyme.nThe elongation factor TAT-SF1 recruits components for splicing. Elongation of transcription, and RNA processing are interconnected/ coupl

26、ed (偶联的偶联的) to ensure the coordination (协同性协同性) of these events.90nProtection from degradation.nIncreased translational efficiency.nTransport to cytoplasm.nSplicing of first intron.91nLinked with the termination of transcription.nThe CTD tail is involved in recruiting the polyadenylation enzymes.nTh

27、e transcribed poly-A signal triggers the reactions.921. Cleavage of the message RNA2. Addition of poly-A3. Termination of transcription9394951. CPSF (cleavage and polyadenylation specificity factor) & CstF (cleavage stimulation factor) bind to the poly-A signal, leading to the RNA cleavage.2. Po

28、ly-A polymerase (PAP) adds 200 As at the 3 end of the RNA, using ATP as a substrate.Fig 12-20 polyadenylation and termination96979899Fig 12-21 Pol I promoter regionUBF 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 transcription100Fig 12-22 Pol III core promoterTFIIIC binds to the promoter, recruiting TFIIIB, which in turn recruits RNAP III1011. RNA polymerases (RNAP, 真核和原核的异同真核和原核的异同) and transcription cycle (Initiation is more complicate, details in bacter