分子细胞生物学前沿

分子细胞生物学专题-转录延伸因子P-TEFb及其活性调控,生命科学学院陈瑞川,细胞生物学博士研究生课程,MainPoints,Transcriptionprocessofeukaryoticgene.P-TEFb:functionsanditsActivityregulation,I.Transcriptionprocess,StepsofclassIIgenetranscription,PICassembly,TFIIA,TFIIB,TFIID,TFIIE,TFIIF,andTFIIHRNApolymeraseII(PolII),TheclassIIpre-initiationcomplex(PIC),RNApolymeraseII(PolII),12subunites:Rpb1-Rpb12About500KdaofMWRpb1:largestsubunit,CTD:Y1S2P3T4S5P6S7Yest:26repeatsC.elegans:32repeatsDrosophila42repeatsMammals:52repeats,Pre-initiation,PICassembly,PIC,PIC,Initiation,Promoterclearance,20-30nt,PolII,CTD,TATA,Pausing,Capping,PolII,CTD,TATA,RNA,Earlyelongation,Capping,Earlyelongation,ProductiveElongation,NELF,P,P,Splicing,ProductiveElongation,Steps:Pre-initiationInitiationPromoterclearanceElongationTermination,Earlyelongation,Pre-initiation,Transcriptioncycle,TranscriptionProcess,P-TEFb,itsfunctionsandactivityregulation,P-TEFb:compositionandproperty,Discoveredin1995.(positivetranscriptionelongationfactorb)AheterodimerofCdk9andcyclinT,includingT1,T2a,T2bandCyclinK.But80%P-TEFbisCDK9/CyclinT1.kinase,phosphorylatesCTDofPolII,DSIFandNELF.,Physiologicalfunctions,AglobaltranscriptionalelongationfactorEssentialformorethan80%mRNAtranscriptionEssentialforembryodevelopment,AdaptedafterPrice,MCB20:2629(2000),P-TEFb在基因转录过程中的作用机制模式图,B.Pathologicalfunctions,ExquisitelyrequiredforHIV-1geneexpressionAbnormallyhighP-TEFbactivitydirectlinktocardiachypertrophyMayalsorelatetotumorprogression,TARRNA,HIVLTR,HIV,P-TEFbisessentialforHIV-1replicationandgeneexpression,Tat,aHIV-1encodedprotein.,Trans-cyclinT1genecausescardiachypertrophy,Trans-vector,Trans-cyclinT1,7SKsnRNA,InactiveP-TEFb-7SKsnRNP,HEXIM1,HEXIM2,LARP7,MePCE,HEXIM1,MePCE,LARP7,7SKsnRNP,5-Cap,7SKsnRNPassemble,ActiveP-TEFbforgeneraltranscription,Brd4,CTD,PolII,mRNA,mRNA,PolII,HEXIM1,7SKRNA,inactiveP-TEFb,RecruitmentofP-TEFbforstimulationoftranscriptionalelongationbybromodomainproteinBrd4(Submittedto:MolCell),HEXIM1,7SKsnRNA,InactiveP-TEFb,ActiveP-TEFb,3/4,1/4,?,Howis7SKsnRNPdisrupted?,HEXIM1,7SKsnRNA,InactiveP-TEFb,PhosphorylatedP-TEFbistaggedforinhibitionthroughassociationwith7SKsnRNA,Howdoescellsignalingcontrolthedisruptionoftheinactivecomplex?,ControlP-TEFbactivitybycalciumsignalingpathway,UVandHMBAcantriggerCa+2influx,Acalcium-dependentsignalingpathwaycontrolsP-TEFbtranscriptionalactivity(inwriting),Inhibitor:,12345,Calciumsignalingpathways,Cellmembrane,L-typechannel,Ca+,Calciumsignalingpathways,CaM-dependentproteinkinases,CaM-dependentproteinphosphatases,CaMKK,CaMKI,CaMKII,CaMKIV,Calcineurin(CaN,PP2B),W7,CsA,FK506,Acalcium-dependentsignalingpathwaycontrolsP-TEFbtranscriptionalactivity(inwriting),Ca+,Hidingandseeking.,Whoisthesecondone?,isoformofPP1catalyticsubunitinvolvesinmediatingthedisruptionof7SKsnRNP,Acalcium-dependentsignalingpathwaycontrolsP-TEFbtranscriptionalactivity(inwriting),Ca+,PP1,PP2B+PP1,?,Invivo:PP2BandPP1workcooperativelytodisrupttheinactivecomplex,Invitro:PP2BandPP1directlydisruptinactivecomplex,PP2B:letmeworkfirst.,Ca+,PP1,Ca+2/PP2BandPP1pathwayscooperativelymediateUVandHMBAinduceddisruptionofinactivecomplex,HowdoPP2BandPP1cooperativelydisruptinactiveP-TFbcomplex?,Invitro.OnlyPP1candephosphorylatepT186ofCDK9,Invitro.PP2BandPP1workcooperativelytodephosphorylatepT186ofCDK9,InactiveP-TEFb,P,P,PP2B,PP1,Twostepsdephosphorylationfrodissociating7SKsnRNP,Ca2+,PP2B,PP1a,CaM,PP2B,PP1a,inactiveP-TEFb,P,P,HEXIM1,Acalciumsignalingpathwaydependenttwo-stepdephosphorylationtoreleaseP-TEFb,P,HEXIM1,pT186,Brd4,HowisliberatedP-TFFbRecruitedtochromatin,ActiveP-TEFbforgeneraltranscription,Brd4,CTD,PolII,mRNA,mRNA,PolII,inactiveP-TEFb,Brd4,?,Dynamicshiftbetweendifferentcomplex.,ModifiedNuclearFractionation,Chromatin-freefraction(LSF),Nucleus,Chromatin-boundfraction(HSF),Nucleus,AlmostallofBrd4associatewithchromatinatbasalstate,StimuationinducesBrd4releasefromchromatin,ThereleaseofBrd4fromchromatinisessentialfr