分子细胞生物学前沿博士.ppt

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,Main Points,Transcription process of eukaryotic gene. P-TEFb: functions and its Activity regulation,I. Transcription process,Steps of class II gene transcription,PIC assembly,TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH RNA polymerase II (Pol II),The class II pre-initiation complex (PIC),RNA polymerase II (Pol II),12 subunites: Rpb1- Rpb12 About 500Kda of MW Rpb1: largest subunit, CTD: Y1S2P3T4S5P6S7 Yest: 26 repeats C. elegans: 32 repeats Drosophila 42 repeats Mammals: 52 repeats,Pre-initiation,PIC assembly,PIC,PIC,Initiation,Promoter clearance,20-30 nt,Pol II,CTD,TATA,Pausing,Capping,Pol II,CTD,TATA,RNA,Early elongation,Capping,Early elongation,Productive Elongation,NELF,P,P,Splicing,Productive Elongation,Steps: Pre-initiation Initiation Promoter clearance Elongation Termination,Early elongation,Pre-initiation,Transcription cycle,Transcription Process,P-TEFb, its functions and activity regulation,P-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 and NELF.,Physiological functions,A global transcriptional elongation factor Essential for more than 80% mRNA transcription Essential for embryo development,Adapted after Price, MCB 20:2629 (2000),P-TEFb在基因转录过程中的作用机制模式图,B. Pathological functions,Exquisitely required for HIV-1 gene expression Abnormally high P-TEFb activity direct link to cardiac hypertrophy May also relate to tumor progression,TAR RNA,HIV LTR,HIV,P-TEFb is essential for HIV-1 replication and gene expression,Tat, a HIV-1 encoded protein.,Trans-cyclin T1 gene causes cardiac hypertrophy,Trans-vector,Trans-cyclin T1,7SK snRNA,Inactive P-TEFb-7SK snRNP,HEXIM1,HEXIM2,LARP7,MePCE,HEXIM1,MePCE,LARP7,7SK snRNP,5-Cap,7SK snRNP assemble,Active P-TEFb for general transcription,Brd4,CTD,Pol II,mRNA,mRNA,Pol II,HEXIM1,7SK RNA,inactive P-TEFb,Recruitment of P-TEFb for stimulation of transcriptional elongation by bromodomain protein Brd4 (Submitted to: Mol Cell),HEXIM1,7SK snRNA,Inactive P-TEFb,Active P-TEFb,3/4,1/4,?,How is 7SK snRNP disrupted?,HEXIM1,7SK snRNA,Inactive P-TEFb,Phosphorylated P-TEFb is tagged for inhibition through association with 7SK snRNA,How does cell signaling control the disruption of the inactive complex?,Control P-TEFb activity by calcium signaling pathway,UV and HMBA can trigger Ca+2 influx,A calcium-dependent signaling pathway controls P-TEFb transcriptional activity (in writing),Inhibitor:,1 2 3 4 5,Calcium signaling pathways,Cell membrane,L-type channel,Ca+,Calcium signaling pathways,CaM-dependent protein kinases,CaM-dependent protein phosphatases,CaMKK, CaMKI, CaMKII, CaMKIV,Calcineurin (CaN, PP2B),W7,CsA, FK506,A calcium-dependent signaling pathway controls P-TEFb transcriptional activity (in writing),Ca+,Hiding and seeking .,Who is the second one?, isoform of PP1 catalytic subunit involves in mediating the disruption of 7SK snRNP,A calcium-dependent signaling pathway controls P-TEFb transcriptional activity (in writing),Ca+,PP1,PP2B + PP1,?,In vivo: PP2B and PP1 work cooperatively to disrupt the inactive complex,In vitro: PP2B and PP1 directly disrupt inactive complex,PP2B: let me work first.,Ca+,PP1,Ca+2/PP2B and PP1 pathways cooperatively mediate UV and HMBA induced disruption of inactive complex,How do PP2B and PP1 cooperatively disrupt inactive P-TFb complex?,In vitro. Only PP1 can dephosphorylate pT186 of CDK9,In vitro. PP2B and PP1 work cooperatively to dephosphorylate pT186 of CDK9,Inactive P-TEFb,P,P,PP2B,PP1,Two steps dephosphorylation fro dissociating 7SK snRNP,Ca2+,PP2B,PP1a,CaM,PP2B,PP1a,inactive P-TEFb,P,P,HEXIM1,A calcium signaling pathway dependent two-step dephosphorylation to release P-TEFb,P,HEXIM1,pT186,Brd4,How is liberated P-TFFb Recruited to chromatin,Active P-TEFb for general transcription,Brd4,CTD,Pol II,mRNA,mRNA,Pol II,inactive P-TEFb,Brd4,?,Dynamic shift between different complex.,Modified Nuclear Fractionation,Chromatin-free fraction (LSF),Nucleus,Chromatin-bound fraction (HSF),Nucleus,Almost all of Brd4 associate with chromatin at basal state,Stimuation induces Brd4 release from chromatin,The release of Brd4 from chromatin is essenti