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1、Transcription and Post-transcription ModificationCentraldogmaWHICHCAMEFIRST,THEchickenortheegg?Thebiologicalsilenceshaveavariation:whichcamefirst,DNAorprotein?Yousee,amongthemanytasksperformedbyproteinsisassemblingDNAmolecules.ButDNAcontainstheinformationneededtomakeproteins.Sowhichcamefirst?RNA and

2、 RNA worldWalterGilbert1980NobelPrizeOrigin-of-LifeTheoriesRNAhastheabilitytoactasbothgenesandenzymesThesynthesisofRNAmoleculesusingDNAstrandsasthetemplatessothatthegeneticinformationcanbetransferredfromDNAtoRNA.Fourstages:Initiation,Elongation,Termination,Post-transcriptionalmodificationTranscripti

3、on Only the template strand is used for the transcription,but the coding strand is not.Both strands can be used as the templates.The transcription direction on different strands is opposite.This feature is referred to as the asymmetric transcription.Asymmetric transcriptionTemplateThe template stran

4、d is the strand from which the RNA is actually transcribed.It is also termed as antisense strand.The coding strand is the strand whose base sequence specifies the amino acid sequence of the encoded protein.Therefore,it is also called as sense strand.Both processes use DNA as the template.Phosphodies

5、ter bonds are formed in both cases.Both synthesis directions are from 5 to 3.Similarity between replication and transcriptionReplicationTranscriptionTemplateDouble strandsSingle strandSubstratedNTPNTPPrimer yesno EnzymeDNA polymeraseRNA polymeraseProductdsDNAssRNABase pairA-T,G-CA-U,T-A,G-CDifferenc

6、es between replication and transcriptionThe whole genome of DNA needs to be replicated,but only small portion of genome is transcribed in response to the development requirement,physiological need and environmental changes.DNA regions that can be transcribed into RNA are called structural genes.What

7、dothemostDNAdoindeed?General concepts of Transcription ProcessThree phases:initiation,elongation,and termination.The prokaryotic RNA-pol can bind to the DNA template directly in the transcription process.The eukaryotic RNA-pol requires co-factors to bind to the DNA template together in the transcrip

8、tion process.Transcription bubbleTranscription in prokaryotesTranscription Unit Optimal PromoterThe35sequenceisusedforinitialrecognition,andthe10sequenceisusedforthemeltingreactionthatconvertsaclosedcomplextoanopencomplex.Duringtranscription,thebubbleismaintainedwithinbacterialRNApolymerase,whichunw

9、indsandrewindsDNA,maintainstheconditionsofthepartnerandtemplateDNAstrands,andsynthesizesRNA.Bacterial RNA Polymerases A single type of RNA polymerase is responsible for almost all synthesis of mRNA,rRNA and tRNA in a eubacterium.About 7,000 RNA polymerase molecules are present in an E.coli cell.Prob

10、ably 2,0005,000 enzymes are synthesizing RNA at any one time,the number depending on the growth conditions.How does RNA polymerase work?RNApolbsubunitisthetargetofRifamycinHow many sigma factors exist in E.coli?How does transcription initiate?Four stages of TranscriptionTerminationThe terminator is

11、in the transcript,not the DNAForms a hairpinSelf-complementaryThe hairpin structure is the signal for terminationRho()-dependent vs.-independent Intrinsic terminators-independentAn inverted repeat that allows a hairpin to form at the end of the transcriptsA string of Ts in the nontemplate strand tha

12、t results in a string of weak rU-dA base pairs holding the transcript to the template strandRhofactorpursuesRNApolymerasealongtheRNAandcancauseterminationwhenitcatchestheenzymepausingatarho-dependentterminator.Termination of-dependentTranscription in EukaryotesRNA polymerases in EukaryotesRNApolymer

13、aseItranscribesrRNARNApolymeraseIItranscribeshnRNA（heterogeneousnuclearRNA）RNApolymeraseIIItranscribestRNAandothersmallRNAs.More than 10 Subunits to form RNA polymerase II Have you ever wondered how your genome works?Well,thanks to scientists like Roger Kornberg,awarded the 2006 Nobel prize for Chem

14、istry,who has painstakingly studied the micromechanics of transcription,were getting a much clearer picture of what happens inside the nucleus,and how the billions of metres of DNA in your body are converted into RNA by an enzyme called RNA polymerase II.-from the announcement of 2006 Nobel prize fo

15、r chemistry Animal RNA Polymerases AnimalDNA-dependentRNAPolymerasesClass -amanitin sensitivity Major Products IInsensitiverRNAIILowConc.(1-10nM)hnRNAIIIHighconc.tRNA,5SRNAandsmallRNAsAmanita phalloides(the death cap)Structure of-amanitinEukaryotic Transcription InitiationTranscription initiation ne

16、eds promoter and upstream regulatory regions.The cis-acting elements are the specific sequences on the DNA template that regulate the transcription of one or more genes.Cis-acting elementRNA-pol does not bind the promoter directly.RNA-pol II associates with six transcription factors,TFII A-TFII H.Th

17、e trans-acting factors are the proteins that recognize and bind directly or indirectly cis-acting elements and regulate its activity.Transcription factors EnhancerEnhancers Work Upstream,Downstream or in the Middle of a Gene They also work forwards or backwardsPossible ways of working Different tran

18、scription factors Order of binding(differing concentrations)Affinity of transcription factors TATA box is a septamer(TATAAAA)at-25 and is involved in positioning the enzyme for correct initiation.CAAT box(CCAATCT)is at 75 and is recognized by a large group of transcription factors and plays a strong

19、 role in determining the efficiency of the promoter.GC box is at-90 contains the sequence GGGCGG and is recognized by the factor SP1.Elements combination in type II Promoters Enhancer;Dehancer;Silencer;Upstream Activating Sequences(UAS)TFIIA activates TBP by relieving the repression that is caused b

20、y the TAFs TFIIB binds adjacent to TBP and TATA boxTFIID is a complex protein containing a TATA-box binding protein and 8-10 TBP-associated factors(TAFs)TBP:TATA-binding protein TAFs:TBP-associated factors TFIIF consists of two subunits.The larger subunit has an ATP-dependent DNA helicase activity a

21、nd the small one contacts the core polymerase.TFIIE and TFIIH are required for promoter clearance to allow RNA polymerase to commence movement away from the promoter.Class II transcription factorspolTFHTAFTFFTAFTAFTFATFBTBPRNA pol with transcription factors form transcription initiation complex.TF I

22、I D is the only factor which can recognize specific sites.TATA DNATATAPOL-TFFABPre initiation complexPOL-TFFHETBP TAFTFD-A-B-DNA complexABTATAHECTD-PCTD tail of RNA pol II is phosphorylated by TFHTBP TAFTBP TAFTBPTAFTBPCTD（Carboxyl Terminal Domain)is repeated sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser

23、TFIIH has several activities,including an ATPase,a helicase,and a kinase activity that can phosphorylate the CTD tail of RNA polymerase II;it is also involved in repair of damage to DNA.Most of the TFII factors are released before RNA polymerase II leaves the promoter.PhosphorylationoftheCTDbythekin

24、aseactivityofTFIIHmaybeneededtoreleaseRNApolymerasetostarttranscription.End of InitiationTranscription unit for RNA polymerase I Promoters in type III gene upstream promoter(type 3)and internal promoter(type1,2)upstream promoter:U6 snRNA Internal promoter:5S RNA and tRNAInitiation in type 1 and 2 ge

25、ne with polymerase III tRNA5S RNA Initiation RNA pol I RNA pol III RNA pol II_ATP requirement no no yes_ A and B or TATA box core consensus sq.core element C box Inr _ CAAT box upstream element UCE GC box etc_ general TFs SL1 TFIIIA B C various TFIIs_ upstream factors UBF various up-stream factors_T

26、BP is a universal factor Transcriptional elongationCTD phosphorylation status of RNA pol IISteps leading to transcriptional activation Promoter escape/clearanceTransition to elongation phaseWhat happens during transcriptional elongation?Original contacts within pre-initiation complex abolished Forma

27、tion of new contacts with elongation factors Phosphorylation of CTD Change of RNA pol II to a ternary complex=high stability CTD consists of heptad repeats of the consensus sequence:YSPTSPS Promoter clearance:Ser#5 gets phosphorylated Transition to elongation:Ser#2 gets phosphorylatedExperimental ev

28、idence for elongation factors Comparison of RNAPII elongation rate in vitro:100-300 nt/min,frequent pauses,and sometimes full arrest in vivo:1200-2000 nt/minWhy the discrepancy?Use of pharmacological agents DRB(5,6-dichloro-1-D-ribofuranosylbenzimidazole DRB,nucleotide-analogue,cause inhibition of h

29、nRNA transcription by arresting RNA pol II in vivo,but not purified RNA pol II.Possible target?These evidence suggest existence of factors that facilitate transcriptional elongation RNA polymerase II often encounters pauses&arrests Arrest(irreversible backsliding 7-14 nts)Pause(back-tracking 2-4 nts

30、)Function of elongation factors:minimize these pauses&arrestsHIV virus can transactivate by hijacking elongation machineryHIV can bypass pre-initiation complex and head straight for elongation by hijacking RNA pol II from hostP-TEFb phosphorylates RNA polI CTDTat:HIVs own elongation factorTerminatio

31、n of Eukaryotic TranscriptionThe termination sequence is AATAAA followed by GT repeats.The termination is closely related to the post-transcriptional modification.TypeIIgenes:TranscriptionstopsafterAATAAA-Polyadenylationsignal.TypeIgenes:3-4consecutiveTsTypeIIIgenes:StopaftersynthesisofserialUs.Meth

32、odology in Gene Transcription Reporter gene transcription Gel mobility shiftDNase FootprintingChIPNuclear run-on transcription ChangepromotersequencePromoterelementsaredefinedbymutationsandfootprintingHow to determine promoter elementsGel Mobility ShiftDNase FootprintingIdentification of Target Gene

33、s by ChIP Promoter ArrayChromatin ImmunoprecipitationPost-transcriptionalProcessingofRNAMakingendsofRNARNAsplicingPrimary Transcript PrimaryTranscript-theinitialmoleculeofRNAproduced-hnRNA（heterogenousnuclearRNA）Inprokaryotes,DNARNAproteinincytoplasmconcurrentlyIneukaryotesnuclearRNACpRNAProcessingofeukaryoticpre-mRNAHumandystrophingenehas79exons,spansover2,300-Kbandrequiresover16hourstobetranscri