分子生物学教学课件：Part 3 Chapter 12 Mechanisms of transcription

1、MOLECULAR BIOLOGY OF GENECh 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 transcription cycleThe transcription cycle in bacteriaTranscription in eukaryotesThe Cen

2、tral DogmaTranscription is very similar to DNA replication but there are some important differences:RNA is made of ribonucleotidesRNA polymerase catalyzes the reactionThe synthesized RNA does not remain base-paired to the template DNA strandLess accurate (error rate: 10-4)Transcription selectively c

3、opies only certain parts of the genome and makes one to several hundred, or even thousand, copies of any given section of the genome. Transcription of DNA into RNATopic 1: RNA Polymerase and The Transcription Cycle7CHAPTER12: Mechanisms of TranscriptionRNA polymerases come in different forms, but sh

4、are many featuresRNA polymerases performs essentially the same reaction in all cellsBacteria have only a single RNA polymerases while in eukaryotic cells there are three: RNA Pol I, II and IIIRNA polymerase and the transcription cycleThe subunits of RNA polymerasesRNA Pol II is the focus of eukaryot

5、ic transcription, because it is the most studied polymerase, and is also responsible for transcribing most 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 gen

6、esThe Bacterial RNA PolymeraseThe core enzyme alone synthesizes RNAaabbwaabwRPB3RPB11RPB2RPB1RPB6prokaryoticeukaryoticb“Crab claw” shape of RNAP (The shape of DNA pol is_)Active center cleftThere are various channels allowing DNA, RNA and ribonucleotides (rNTPs) into and out of the enzymes active ce

7、nter cleftTranscription by RNA polymerase proceeds in a series of stepsInitiationElongationTerminationRNA polymerase and the transcription cycleInitiationPromoter: the DNA sequence that initially binds the RNA polymerase The structure of promoter-polymerase complex undergoes structural changes to pr

8、oceed transcription DNA at the transcription site unwinds and a “bubble” formsDirection of RNA synthesis occurs in a 5-3 direction (3-end growing)Binding (closed complex)Promoter “melting” (open complex)Initial transcriptionElongationOnce the RNA polymerase has synthesized a short stretch of RNA ( 1

9、0 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 growing RNA chain TerminationAfter the p

10、olymerase 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.Elongation and terminationTerminationElongationTranscri

11、ption initiation involves 3 defined stepsForming closed complexForming open complexPromoter escapeRNA polymerase and the transcription cycle The initial binding of polymerase to a promoter DNA remains double strandedThe enzyme is bound to one face of the helixClosed complexOpen Complexthe DNA strand

12、 separate over a distance of 14 bp (-11 to +3 ) around the start site (+1 site)Replication bubble forms Stable ternary complexThe enzyme escapes from the promoterThe transition to the elongation phase Stable ternary complex =DNA +RNA + enzymeBinding (closed complex)Promoter “melting” (open complex)I

13、nitial transcriptionElongation and terminationTerminationElongationTopic 2The transcription cycle in bacteriaCHAPTER12: Mechanisms of Transcription2-1:RNA Polymerase StructureSeparation of s-factor from Core E. coli RNA Polymerase by Phosphocellulose Chromatography Holoenzyme=factor + core enzyme Si

14、gma as a Specificity FactorCore enzyme without the s subunit could not transcribe viral DNA, yet had no problems with highly nicked calf thymus DNAWith s subunit, the holoenzyme worked equally well on both types of DNATesting TranscriptionBautz and colleagues demonstrated this by hybridizing the lab

15、eled product of the holoenzyme or the core enzyme to authentic T4 phage RNA and then checking for RNase resistance.Testing TranscriptionIn fact, Bautz and associates found that about 30% of the labeled RNA made by the core polymerase in vitro became RNase-resistant after hybridization to authentic T

16、4 RNA. Thus, the core enzyme acts in an unnatural way by transcribing both DNA strands.Summary: The key player in the transcription process is RNA polymerase. The E. coli is composed of a core, which contains the basic transcription machinery, and a s-factor, which directs the core to transcribe spe

17、cific gene.2-2:Bacterial promoters vary in strength and sequences, but have certain defining features1. Promoter2. Binding of RNA Polymerase to Promoters3. Promoter StructurePromotersNicks and gaps are good sites for RNA polymerase to bind nonspecificallyPresence of the s-subunit permitted recogniti

18、on of authentic RNA polymerase binding sitesPolymerase binding sites are called promotersTranscription that begins at promoters is specific, directed by the s-subunit3H T7 phage DNAHolo or coreMore T7 phage DNAHow does s change the way the core polymerase behaves toward promoters? David Hinkle and M

19、ichael Chamberlin used nitrocellulose filter-binding studies to help answer this question. Binding of RNA Polymerase to PromotersTemperature and RNA Polymerase BindingAs temperature is lowered, the binding of RNA polymerase to DNA decreases dramaticallyHigher temperature promotes DNA meltingRNA Poly

20、merase BindingHinkle and Chamberlin proposed:RNA polymerase holoenzyme binds DNA loosely at firstBinds at promoter initially or Scans along the DNA until it finds oneComplex with holoenzyme loosely bound at the promoter is a closed promoter complex as DNA is in a closed ds formHoloenzyme can then me

21、lt a short DNA region at the promoter to form an open promoter complex with polymerase bound tightly to DNARNA Polymerase/Promoter BindingThe 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

22、stretch of 17-19 nt. Position +1 is the transcription start site.Promoters recognized by E. coli s factorSummary: The s-factor allows initiation of transcription by the RNA polymerase holoenzyme to bind tightly to a promoter. Prokaryotic promoters contain two regions centered at -10 and -35 bp upstr

23、eam of the transcription start site.Consensus sequence of the -35 and -10 regionCore Promoter ElementsThere is a region common to bacterial promoters described as 6-7 bp centered about 10 bp upstream of the start of transcription = -10 boxAnother short sequence centered 35 bp upstream is known as th

24、e -35 boxComparison of thousands of promoters has produced a consensus sequence for each of these boxesPromoter StrengthConsensus sequences:-10 box sequence approximates TAtAaT-35 box sequence approximates TTGACaMutations that weaken promoter binding:Down mutationsIncrease deviation from the consens

25、us sequenceMutations that strengthen promoter binding:Up mutationsDecrease deviation from the consensus sequenceConfers additional specificityUP-element is an additional DNA elements that increases polymerase binding by providing the additional interaction site for RNA polymeraseUP ElementFig 12-5c:

26、 bacterial promoterAnother class of s70 promoter lacks a 35 region and has an “extended 10 element” compensating for the absence of 35 regionFig 12-5d: bacterial promoterAn additional DNA element that binds RNA polymerase has been found just downstream from the -10 element. This new element I called

27、 the discriminator. The strength of the interaction between the discriminator and polymerase influences the stability of the complex between the enzyme and the promoter.Summary: Bacterial promoters contain two regions centered at -10 and -35bp upstream of the transcription start site. In E.coli, the

28、se bear a greater or lesser resemblance to two consensus sequences: TATAAT and TTGACA, respectively. In general, the more closely regions within a promoter resemble these consensus sequences, the stronger that promoter will be. Some extraordinarily strong promoters contain an extra element upstream

29、of the core promoter. This makes these promoters even more attractive to RNA polymerase.2-3 Transcription InitiationUntil 1980, it was a common assumption that transcription initiation ended when RNA polymerase formed the first phosphodiester bond, joining the first two nucleotides in the growing RN

30、A chain. Gralla reported that initiation is more complex than that.Carpousis and Gralla found that very small oligonucleotides (2-6 nt long) are made without RNA polymerase leaving the DNAAbortive transcripts such as these have been found up to 10 ntE. coli RNA polymerase DNA bearing a mutant E. col

31、i lac promoter known as the lac UV5 promoterIncubate with heparinSynthesis of short oligonucleotides by RNApolymerase bound to a promoter.Mechanism of initial transcription瞬时漂移蠕虫移动蜷缩Stages of Transcription InitiationFormation of a closed promoter complexConversion of the closed promoter complex to a

32、n open promoter complexPolymerizing the early nucleotides polymerase at the promoterPromoter clearance transcript becomes long enough to form a stable hybrid with templateThe Functions of sGene selection for transcription by s causes tight binding between RNA polymerase and promotersTight binding de

33、pends on local melting of DNA that permits open promoter complexDissociation of s from core after sponsoring polymerase-promoter bindingexpect s to stimulate initiation of transcription?Travers and Burgess: took advantage of the fact that the first nucleotide incorporated into an RNA retains all thr

34、ee of its phosphates (a, b, and g), whereas all other nucleotides retain only their a-phosphate.These investigators incubated polymerase core in the presence of increasing amounts of s in two separate sets of reactions. In some reactions, the labeled nucleotide was 14CATP, which is incorporated thro

35、ughout the RNA and therefore measures elongation, as well as initiation, of RNA chains. In the other reactions, the labeled nucleotide was g-32PATP or g-32PGTP, whose label should be incorporated only into the first position of the RNA, and therefore is a measure of transcription initiation.Sigma St

36、imulates Transcription InitiationSigma Stimulates Transcription InitiationStimulation by s appears to cause both initiation and elongationOr stimulating initiation provides more initiated chains for core polymerase to elongateReuse of sDuring initiation s can be recycled for additional use in a proc

37、ess called the s cycleCore enzyme can release s which then associates with another core enzymeTravers and Burgess allowed RNA polymerase holoenzyme to initiate and elongate RNA chains on a T4 DNA template at low ionic strength, so the polymerases could not dissociate from the template to start new R

38、NA chains. The red curve shows the initiation of RNA chains, measured by g-32PATP and g-32PGTP incorporation, under these conditions. After 10 min (arrow), when most chain initiation had ceased, the investigators added new, rifampicin-resistant core polymerase in the presence (green) or absence (blu

39、e) of rifampicin.the new core associated with s that had been associated with the original core.the core, not the s, determines rifampicin resistance or sensitivity. In the same 1969 paper, Travers and Burgess demonstrated that s can be recycled The elongating polymerase is a processive machine that

40、 synthesizes and proofreads RNA The transcription cycle in bacteriaDNA enters the polymerase between the pincersStrand separation in the catalytic cleft（催化裂缝）NTP 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 a

41、nnealing in behindSynthesizing by RNA polymerasePyrohosphorolytic （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. T

42、his is stimulated by Gre factor, a elongation stimulation factor.Proofreading by RNA polymeraseTranscription 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

43、, also called intrinsic (内在) terminator The transcription cycle in bacteriaTermination of TranscriptionWhen the polymerase reaches a terminator at the end of a gene it falls off the template and releases the RNAThere are 2 main types of terminatorsIntrinsic terminators function with the RNA polymera

44、se by itself without help from other proteinsOther type depends on auxiliary factor called r, these are r-dependent terminators Rho-Independent TerminationIntrinsic or r-independent termination depends on terminators of 2 elements:Inverted repeat followed immediately byT-rich region in nontemplate s

45、trand of the geneAn inverted repeat predisposes a transcript to form a hairpin structureRho-independent terminator contains a short inverted repeat (20 bp) and a stretch of 8 A:T base pairs. 68Inverted Repeats and HairpinsThe repeat at right is symmetrical around its center shown with a dotA transcr

46、ipt of this sequence is self-complementaryBases can pair up to form a hairpin as seen in the lower panel Structure of an Intrinsic TerminatorA: normal terminatorB: TTTTTTTT to TTTTGCAAModel of Intrinsic TerminationBacterial terminators act by:Base-pairing of something to the transcript to destabiliz

47、e RNA-DNA hybridCauses hairpin to formCausing the transcription to pauseCauses a string of Us to be incorporated just downstream of hairpinWeakest base pairing: A:U make the dissociation easierRho-Dependent TerminationRho caused depression of the ability of RNA polymerase to transcribe phage DNAs in

48、 vitroThis depression was due to termination of transcriptionAfter termination, polymerase must reinitiate to begin transcribing againInitiationElongationRho Affects Chain Elongation, But Not InitiationRoberts allowed E. coli RNA polymerase to transcribe l phage DNA in the presence of increasing con

49、centrations of rho. He used g-32PGTP to measure initiation (red) and 3HUTP to measure elongation (green). Rho depressed the longation rate, but not initiation. (Source: Adapted from Nature 224:116874, 1969.)Rho Affects Chain ElongationThere is little effect of r on transcription initiation, if anyth

50、ing it is increasedThe effect of r on total RNA synthesis is a significant decreaseThis is consistent with action of r to terminate transcription forcing time-consuming reinitiationRho reduces the size of the RNA product(a) Roberts allowed E. coli RNA polymerase to transcribe l DNA in the absence of

51、 rho. He included 3HUTP in the reaction to label the RNA. Finally, he used ultracentrifugation to separate the transcripts by size. He collected fractions from the bottom of the centrifuge tube, so low numbered fractions, at left, contained the largest RNAs. (b) Roberts used E. coli RNA polymerase t

52、o transcribe l DNA in the presence of rho. He also included 14CATP to label the transcripts, plus the 3HlabeledRNA from panel (a). Again, he ultracentrifuged the transcripts to separate them by size. The 14C-labeled transcripts (red) made in the presence of rho were found near the top of the gradient (at right), indicating that they were relatively small. On the other hand, the 3H-labeled transcripts (blue) from the reaction lacking rho were relatively large and the same size as they were originally. Thus, rho has