分子細(xì)胞生物學(xué)前沿ppt課件

1、分子細(xì)胞生物學(xué)專(zhuān)題-轉(zhuǎn)錄延伸因子P-TEFb及其活性調(diào)控生命科學(xué)學(xué)院陳瑞川細(xì)胞生物學(xué)博士研討生課程 Main PointsTranscription process of eukaryotic gene.P-TEFb: functions and its Activity regulationI. Transcription processSteps of class II gene transcriptionPromotor clearenceInitiationPIC assemblyTerminationElongationTFIIA, TFIIB, TFIID, TFIIE, TFII

2、F, and TFIIHRNA polymerase II (Pol II)The class II pre-initiation complex (PIC)RNA polymerase II (Pol II)12 subunites: Rpb1- Rpb12About 500Kda of MWRpb1: largest subunit, CTD: Y1S2P3T4S5P6S7Yest: 26 repeatsC. elegans: 32 repeatsDrosophila 42 repeatsMammals: 52 repeatsPre-initiationPol IICTDPIC assem

3、blyIIFIIHIIEPICPol IICTDIIFPol IICTDIIFII DII AIIBIIHIIE+1DNA tempTATAPICPol IICTDII DII AIIBIIFIIHIIEInitiation55555Pol II55555+1DNA tempTATAPromoter clearance20-30 ntPol IICTD55555DNA tempTATAIISDSIFNELFMTPausingCappingPol IICTD55555DNA tempTATAIISRNADSIFNELFEarly elongation5-triphosphataseguanylt

4、ransferaseNCNCmethyltransferaseMTMTNCNCCappingEarly elongationDNA tempProductiveElongationCycT1CDK9P-TEFbRNACycT1CDK9P-TEFbPol IICTD55555IISDSIF2222CycT1CDK9P-TEFbNELFPol IICTD55555IISDSIFCapped short mRNA2222PPCycT1CDK9P-TEFbSplicing55555Pol II22222RNADNA tempRNAU snRNPProductive ElongationSplicing

5、CycT1CDK9P-TEFbFCP1Pol IIPol IICTD25555222Pol II25555222+15555Pol II5 capRNA55552222Pol IICTDTFIIHNFkBSp1NFATPol IISteps: Pre-initiationInitiationPromoter clearanceElongationTermination InitiationEarly elongationPromoter clearanceElongationTerminationPre-initiationTranscription cycleTranscription Pr

6、ocessP-TEFb, its functions and activity regulationP-TEFb: composition and property Discovered in 1995. (positive transcription elongation factor b) A heterodimer of Cdk9 and cyclin T, including T1, T2a, T2b and Cyclin K. But 80% P-TEFb is CDK9/Cyclin T1. kinase, phosphorylates CTD of Pol II, DSIF an

7、d NELF.CycT1Cdk9Physiological functionsA global transcriptional elongation factorEssential for more than 80% mRNA transcription Essential for embryo developmentCTDPol IIRNANELFNuc失敗轉(zhuǎn)錄CycT1Cdk9P-TEFb勝利轉(zhuǎn)錄RNAPPPPPPPPCycT1Cdk9Adapted after Price, MCB 20:2629 (2000)P-TEFb在基因轉(zhuǎn)錄過(guò)程中的作用機(jī)制方式圖B. Pathological f

8、unctionsExquisitely required for HIV-1 gene expression Abnormally high P-TEFb activity direct link to cardiac hypertrophy May also relate to tumor progressionTAR RNACTDPol II5555CTDPol II5555Abortive TranscriptionCTDPol II5555Short RNACycT1CDK9TatCycT1CDK9TatHIV LTR+1TatTatCycT1CDK9TatCycT1CDK9TatCy

9、cT1CDK9Tat2222HIVP-TEFb is essential for HIV-1 replication and gene expressionTat, a HIV-1 encoded protein. Trans-cyclin T1 gene causes cardiac hypertrophyTrans-vectorTrans-cyclin T1CycT1Cdk97SK snRNAInactive P-TEFb-7SK snRNPHEXIM1HEXIM2LARP7MePCEHEXIM1LARP7Methyl donorCycT1CDK9HEXIM1CycT1CDK9HEXIM1

10、MePCEMePCELARP7CycT1CDK9HEXIM1MePCE7SKLARP75-Cap3-tailMePCE3-tailcapped 7SK7SK5-Cap3-tailTrinity compled7SK snRNP3-tailNascent 7SK5-termini5-CapCycT1CDK9HEXIM17SK snRNP assembleActive P-TEFb for general transcriptionCycT1Cdk9Brd4PPPPPPPPCycT1Cdk9Brd4CTDPol IImRNAmRNAPol IICycT1Cdk9HEXIM17SK RNACycT1

11、Cdk9Brd4Stress/hypertrophic signals/estrogen/ HIV infectioninactiveP-TEFbCycT1Cdk9Brd4activeP-TEFbRecruitment of P-TEFb for stimulation of transcriptional elongation by bromodomain protein Brd4 (Submitted to: Mol Cell) CycT1Cdk9Brd4CycT1Cdk9HEXIM17SK snRNAInactive P-TEFbActive P-TEFbCycT1Cdk9PPP3/41

12、/4?How is 7SK snRNP disrupted?CycT1Cdk9HEXIM17SK snRNAInactive P-TEFbPPPPPPPPPPPPPPPPPhosphorylated P-TEFb is tagged for inhibition through association with 7SK snRNAPP1 treatmentOrStress: Act D, DRB, UVDephosphorylationActive P-TEFbCycT1Cdk9HEXIM1+PPPPPPPPhosphorylationATPHow does cell signaling co

13、ntrol the disruption of the inactive complex?Control P-TEFb activity by calcium signaling pathwayUV and HMBA can trigger Ca+2 influxF1C2 cellSignal pathway inhibitorsCycT1Cdk9HEXIM17SK snRNPInactive P-TEFbPPPPPPPPPPPPPPPPStress treatmentCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+A calcium-dependent signali

14、ng pathway controls P-TEFb transcriptional activity (in writing)Inhibitor:UV:HEXIM1CDK9-f1 2 3 4 5Calcium signaling pathwaysCa+InfluxCycT1Cdk9HEXIM17SK snRNPInactive P-TEFbPPPPPPPPPPPPPPPPCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+Cell membraneL-type channelF1C2 cellCalciumCycT1Cdk9HEXIM17SK snRNPInactive

15、P-TEFbPPPPPPPPPPPPPPPPStress treatmentCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+F1C2 cellCa+InfluxCalcium signaling pathwaysF1C2 cellCa+InfluxCaM-dependent protein kinasesCaMCaM-dependent protein phosphatasesCaMKK, CaMKI,CaMKII, CaMKIVCalcineurin (CaN, PP2B)W7CsA, FK506F1C2 cellW7, CsA, FK506CycT1Cdk9HEXI

16、M17SK snRNPInactive P-TEFbPPPPPPPPPPPPPPPPStress treatmentCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+A calcium-dependent signaling pathway controls P-TEFb transcriptional activity (in writing)F1C2 cellCa+InfluxCaMPP2BHiding and seeking .Who is the second one? isoform of PP1 catalytic subunit involves in me

17、diating the disruption of 7SK snRNPF1C2 cellCycT1Cdk9HEXIM17SK snRNPInactive P-TEFbPPPPPPPPPPPPPPPPCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+A calcium-dependent signaling pathway controls P-TEFb transcriptional activity (in writing)F1C2 cellCa+InfluxCaMPP2BPP1PP2B + PP1?In vivo:PP2B and PP1 work cooperati

18、vely to disrupt the inactive complexIn vitro: PP2B and PP1 directly disrupt inactive complex PP2B:let me work first.F1C2 cellCalciumCycT1Cdk9HEXIM17SK snRNPInactive P-TEFbPPPPPPPPPPPPPPPPStress treatmentCycT1Cdk9Active P-TEFbPPPPPPPHEXIM1+F1C2 cellCa+InfluxCaMPP2BPP1Ca+2/PP2B and PP1 pathways cooper

19、atively mediate UV and HMBA induced disruption of inactive complexHow do PP2B and PP1 cooperatively disrupt inactive P-TFb complex?In vitro.Only PP1 can dephosphorylate pT186 of CDK9In vitro.PP2B and PP1 work cooperatively to dephosphorylate pT186 of CDK9CycT1Cdk9Inactive P-TEFbPPHEXIM17SK snRNAPP2B

20、PP1Two steps dephosphorylation fro dissociating 7SK snRNPCycT1Cdk9plasma membraneLTCCCa2+PP2BnucleusPP1aCaMPP2BPP1ainactiveP-TEFbCycT1Cdk9UV or HMBAPPHEXIM1A calcium signaling pathway dependent two-step dephosphorylation to release P-TEFbCycT1Cdk9?PHEXIM1pT186Brd4How is liberated P-TFFb Recruited to

21、 chromatinActive P-TEFb for general transcriptionCycT1Cdk9Brd4PPPPPPPPCycT1Cdk9Brd4CTDPol IImRNAmRNAPol IICycT1Cdk9HEXIM17SK RNACycT1Cdk9Brd4Stress/hypertrophic signals/estrogen/ HIV infectioninactiveP-TEFbBrd4CycT1Cdk9Brd4?Dynamic shift between different complex.CycT1Cdk9activeP-TEFbHEXIM1CycT1Cdk9activeP-TEFb1/4Modified Nuclear FractionationChromatin-freefraction (LSF)NucleusLow-salt bufferextractionHigh-salt bufferextractionChromatin-bou