分子细胞生物学前沿博士

1、会计学1分子细胞生物学前沿博士分子细胞生物学前沿博士第1页/共67页I. Transcription process第2页/共67页Promotor clearenceInitiationPIC assemblyTerminationElongation第3页/共67页 TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH RNA polymerase II (Pol II)The class II pre-initiation complex (PIC)第4页/共67页12 subunites: Rpb1- Rpb12About 500Kda of MW

2、Rpb1: largest subunit, CTD: Y1S2P3T4S5P6S7 Yest: 26 repeats C. elegans: 32 repeats Drosophila 42 repeats Mammals: 52 repeats第5页/共67页Pre-initiationPol IICTDPIC assemblyIIFIIHIIEPICPol IICTDIIFPol IICTDIIFII DII AIIBIIHIIE+1DNA tempTATA第6页/共67页PICPol IICTDII DII AIIBIIFIIHIIEInitiation55555Pol II55555

3、+1DNA tempTATAPromoter clearance20-30 nt第7页/共67页Pol IICTD55555DNA tempTATAIISDSIFNELFMTPausingCapping第8页/共67页Pol IICTD55555DNA tempTATAIISRNADSIFNELFEarly elongation5-triphosphataseguanyltransferaseNCNCmethyltransferaseMTMTNCNCCapping第9页/共67页Early elongationDNA tempProductiveElongationCycT1CDK9P-TEF

4、bRNACycT1CDK9P-TEFbPol IICTD55555IISDSIF2222CycT1CDK9P-TEFbNELFPol IICTD55555IISDSIFCapped short mRNA2222PPCycT1CDK9P-TEFb第10页/共67页Splicing55555Pol II22222RNADNA tempRNAU snRNPProductive ElongationSplicing第11页/共67页CycT1CDK9P-TEFbFCP1Pol IIPol IICTD25555222Pol II25555222+15555Pol II5 capRNA55552222Po

5、l IICTDTFIIHNFk kBSp1NFATPol II第12页/共67页 Steps: Pre-initiation Initiation Promoter clearance Elongation Termination InitiationEarly elongationPromoter clearanceElongationTerminationPre-initiationTranscription cycleTranscription Process第13页/共67页P-TEFb, its functions and activity regulation第14页/共67页ki

6、nase, phosphorylates CTD of Pol II, DSIF and NELF.CycT1Cdk9第15页/共67页Physiological functionsA global transcriptional elongation factorEssential for more than 80% mRNA transcription Essential for embryo development第16页/共67页CTDPol IIRNANELFNuc失败转录失败转录CycT1Cdk9P-TEFb成功转录成功转录RNAPPPPPPPPCycT1Cdk9Adapted a

7、fter Price, MCB 20:2629 (2000)P-TEFb在基因转录过程中的作用机制在基因转录过程中的作用机制模式图模式图第17页/共67页B. Pathological functionsExquisitely required for HIV-1 gene expression Abnormally high P-TEFb activity direct link to cardiac hypertrophy May also relate to tumor progression第18页/共67页TAR RNACTDPol II5555CTDPol II5555Aborti

8、ve TranscriptionCTDPol II5555Short RNACycT1CDK9TatCycT1CDK9TatHIV LTR+1TatTatCycT1CDK9TatCycT1CDK9TatCycT1CDK9Tat2222HIVP-TEFb is essential for HIV-1 replication and gene expressionTat, a HIV-1 encoded protein. 第19页/共67页Trans-cyclin T1 gene causes cardiac hypertrophyTrans-vectorTrans-cyclin T1第20页/共

9、67页CycT1Cdk97SK snRNAInactive P-TEFb-7SK snRNPHEXIM1HEXIM2LARP7MePCEHEXIM1第21页/共67页LARP7Methyl donorCycT1CDK9HEXIM1CycT1CDK9HEXIM1MePCEMePCELARP7CycT1CDK9HEXIM1MePCE7SKLARP75-Cap3-tailMePCE3-tailcapped 7SK7SK5-Cap3-tailTrinity compled7SK snRNP3-tailNascent 7SK5-termini5-CapCycT1CDK9HEXIM17SK snRNP a

10、ssemble第22页/共67页Active P-TEFb for general transcriptionCycT1Cdk9Brd4第23页/共67页PPPPPPPPCycT1Cdk9Brd4CTDPol IImRNAmRNAPol IICycT1Cdk9HEXIM17SK RNACycT1Cdk9Brd4Stress/hypertrophic signals/estrogen/ HIV infectioninactiveP-TEFbCycT1Cdk9Brd4activeP-TEFbRecruitment of P-TEFb for stimulation of transcription

11、al elongation by bromodomain protein Brd4 (Submitted to: Mol Cell) CycT1Cdk9Brd4第24页/共67页CycT1Cdk9HEXIM17SK snRNAInactive P-TEFbActive P-TEFbCycT1Cdk9PPP3/41/4?How is 7SK snRNP disrupted?第25页/共67页CycT1Cdk9HEXIM17SK snRNAInactive P-TEFbPPPPPPPPPPPPPPPPPhosphorylated P-TEFb is tagged for inhibition th

12、rough association with 7SK snRNAPP1 treatmentOrStress: Act D, DRB, UVDephosphorylationActive P-TEFbCycT1Cdk9HEXIM1+PPPPPPPPhosphorylationATP第26页/共67页第27页/共67页第28页/共67页第29页/共67页F1C2 cellSignal pathway inhibitorsCycT1Cdk9HEXIM17SK snRNPInactive P-TEFbPPPPPPPPPPPPPPPPStress treatmentCycT1Cdk9Active P-T

13、EFbPPPPPPPHEXIM1+A calcium-dependent signaling pathway controls P-TEFb transcriptional activity (in writing)Inhibitor: UV:HEXIM1CDK9-f1 2 3 4 5第30页/共67页Ca+InfluxCycT1Cdk9HEXIM17SK snRNPInactive P-TEFbPPPPPPPPPPPPPPPPCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+Cell membraneL-type channel第31页/共67页F1C2 cellCal

14、ciumCycT1Cdk9HEXIM17SK snRNPInactive P-TEFbPPPPPPPPPPPPPPPPStress treatmentCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+F1C2 cellCa+Influx第32页/共67页F1C2 cellCa+InfluxCaM-dependent protein kinasesCaMCaM-dependent protein phosphatasesCaMKK, CaMKI,CaMKII, CaMKIVCalcineurin (CaN, PP2B)W7CsA, FK506第33页/共67页F1C2 ce

15、llW7, CsA, FK506CycT1Cdk9HEXIM17SK snRNPInactive P-TEFbPPPPPPPPPPPPPPPPStress treatmentCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+A calcium-dependent signaling pathway controls P-TEFb transcriptional activity (in writing)F1C2 cellCa+InfluxCaMPP2B第34页/共67页Who is the second one?第35页/共67页第36页/共67页F1C2 cellCyc

16、T1Cdk9HEXIM17SK snRNPInactive P-TEFbPPPPPPPPPPPPPPPPCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+A calcium-dependent signaling pathway controls P-TEFb transcriptional activity (in writing)F1C2 cellCa+InfluxCaMPP2BPP1PP2B + PP1?第37页/共67页第38页/共67页第39页/共67页第40页/共67页F1C2 cellCalciumCycT1Cdk9HEXIM17SK snRNPInacti

17、ve P-TEFbPPPPPPPPPPPPPPPPStress treatmentCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+F1C2 cellCa+InfluxCaMPP2BPP1Ca+2/PP2B and PP1 pathways cooperatively mediate UV and HMBA induced disruption of inactive complex第41页/共67页第42页/共67页第43页/共67页第44页/共67页CycT1Cdk9Inactive P-TEFbPPHEXIM17SK snRNAPP2BPP1Two steps de

18、phosphorylation fro dissociating 7SK snRNP第45页/共67页CycT1Cdk9plasma membraneLTCCCa2+PP2BnucleusPP1a aCaMPP2BPP1a ainactiveP-TEFbCycT1Cdk9UV or HMBAPPHEXIM1A calcium signaling pathway dependent two-step dephosphorylation to release P-TEFbCycT1Cdk9?PHEXIM1pT186Brd4第46页/共67页第47页/共67页Active P-TEFb for ge

19、neral transcriptionCycT1Cdk9Brd4第48页/共67页PPPPPPPPCycT1Cdk9Brd4CTDPol IImRNAmRNAPol IICycT1Cdk9HEXIM17SK RNACycT1Cdk9Brd4Stress/hypertrophic signals/estrogen/ HIV infectioninactiveP-TEFbBrd4CycT1Cdk9Brd4?Dynamic shift between different complex.CycT1Cdk9activeP-TEFbHEXIM1CycT1Cdk9activeP-TEFb1/4第49页/共

20、67页Chromatin-freefraction (LSF)NucleusLow-salt bufferextractionHigh-salt bufferextractionChromatin-boundfraction (HSF)Nuclear lysisfraction (NLF)NucleusNucleus第50页/共67页第51页/共67页第52页/共67页第53页/共67页第54页/共67页第55页/共67页CycT1Cdk9ProductiveelongationHEXIM1+222 2PP2B+PP1Kinase?T186CycT1Cdk9PBrd4mRNAPol II555

21、 5AcAcHDACRecruitP-TEFb topromotermRNAPol IIH4 tail555 5H4K5/8deacetylatedSignalDeacetylateH4K5/8AcmRNAPol IIAcAcBrd4Acetyl-nucleosome555 5CTDReleaseBrd4 fromchromatin7SKT1867SKCycT1Cdk9PHEXIM17SK snRNPAi_Fig7CycT1Cdk9PAdaptorBrd4Brd4第56页/共67页HDACSignal?CycT1Cdk9KinaseT186CycT1Cdk9P?第57页/共67页Thank y

22、ou!第58页/共67页CycT1CDK9P-TEFbFCP1Pol IIPol IICTD25555222Pol II25555222+15555Pol II5 capRNA55552222Pol IICTDTFIIHNFk kBSp1NFATPol II第59页/共67页Physiological functionsA global transcriptional elongation factorEssential for more than 80% mRNA transcription Essential for embryo development第60页/共67页CTDPol IIRNANELFNuc失败转录失败转录CycT1Cdk9P-TEFb成功转录成功转录RNAPPPPPPPPCycT1Cdk9Adapted after Price, MCB 20:2629 (2000)P-TEFb在基因转录过程中的作用机制在基因转录过程