分子細胞生物學前沿博士

1、會計學1分子細胞生物學前沿博士分子細胞生物學前沿博士第1頁/共67頁I. Transcription process第2頁/共67頁Promotor clearenceInitiationPIC assemblyTerminationElongation第3頁/共67頁 TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH RNA polymerase II (Pol II)The class II pre-initiation complex (PIC)第4頁/共67頁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頁/共67頁Pre-initiationPol IICTDPIC assemblyIIFIIHIIEPICPol IICTDIIFPol IICTDIIFII DII AIIBIIHIIE+1DNA tempTATA第6頁/共67頁PICPol IICTDII DII AIIBIIFIIHIIEInitiation55555Pol II55555

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

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

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

6、nase, phosphorylates CTD of Pol II, DSIF and NELF.CycT1Cdk9第15頁/共67頁Physiological functionsA global transcriptional elongation factorEssential for more than 80% mRNA transcription Essential for embryo development第16頁/共67頁CTDPol IIRNANELFNuc失敗轉錄失敗轉錄CycT1Cdk9P-TEFb成功轉錄成功轉錄RNAPPPPPPPPCycT1Cdk9Adapted a

7、fter Price, MCB 20:2629 (2000)P-TEFb在基因轉錄過程中的作用機制在基因轉錄過程中的作用機制模式圖模式圖第17頁/共67頁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頁/共67頁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頁/共67頁Trans-cyclin T1 gene causes cardiac hypertrophyTrans-vectorTrans-cyclin T1第20頁/共

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

10、ssemble第22頁/共67頁Active P-TEFb for general transcriptionCycT1Cdk9Brd4第23頁/共67頁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頁/共67頁CycT1Cdk9HEXIM17SK snRNAInactive P-TEFbActive P-TEFbCycT1Cdk9PPP3/41/4?How is 7SK snRNP disrupted?第25頁/共67頁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頁/共67頁第27頁/共67頁第28頁/共67頁第29頁/共67頁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頁/共67頁Ca+InfluxCycT1Cdk9HEXIM17SK snRNPInactive P-TEFbPPPPPPPPPPPPPPPPCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+Cell membraneL-type channel第31頁/共67頁F1C2 cellCal

14、ciumCycT1Cdk9HEXIM17SK snRNPInactive P-TEFbPPPPPPPPPPPPPPPPStress treatmentCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+F1C2 cellCa+Influx第32頁/共67頁F1C2 cellCa+InfluxCaM-dependent protein kinasesCaMCaM-dependent protein phosphatasesCaMKK, CaMKI,CaMKII, CaMKIVCalcineurin (CaN, PP2B)W7CsA, FK506第33頁/共67頁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頁/共67頁Who is the second one?第35頁/共67頁第36頁/共67頁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頁/共67頁第38頁/共67頁第39頁/共67頁第40頁/共67頁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頁/共67頁第42頁/共67頁第43頁/共67頁第44頁/共67頁CycT1Cdk9Inactive P-TEFbPPHEXIM17SK snRNAPP2BPP1Two steps de

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

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

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

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

22、ou!第58頁/共67頁CycT1CDK9P-TEFbFCP1Pol IIPol IICTD25555222Pol II25555222+15555Pol II5 capRNA55552222Pol IICTDTFIIHNFk kBSp1NFATPol II第59頁/共67頁Physiological functionsA global transcriptional elongation factorEssential for more than 80% mRNA transcription Essential for embryo development第60頁/共67頁CTDPol IIRNANELFNuc失敗轉錄失敗轉錄CycT1Cdk9P-TEFb成功轉錄成功轉錄RNAPPPPPPPPCycT1Cdk9Adapted after Price, MCB 20:2629 (2000)P-TEFb在基因轉錄過程中的作用機制在基因轉錄過程