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(zhuǎn)錄失敗轉(zhuǎn)錄CycT1Cdk9P-TEFb成功轉(zhuǎn)錄成功轉(zhuǎn)錄RNAPPPPPPPPCycT1Cdk9Adapted a

7、fter Price, MCB 20:2629 (2000)P-TEFb在基因轉(zhuǎn)錄過程中的作用機制在基因轉(zhuǎn)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(zhuǎn)錄失敗轉(zhuǎn)錄CycT1Cdk9P-TEFb成功轉(zhuǎn)錄成功轉(zhuǎn)錄RNAPPPPPPPPCycT1Cdk9Adapted after Price, MCB 20:2629 (2000)P-TEFb在基因轉(zhuǎn)錄過程中的作用機制在基因轉(zhuǎn)錄過程