Biochemische und strukturbiologische Analyse der Regulation des humanen Transkriptionselongationsfaktors P-TEFb

人转录延伸因子 P-TEFb 调控的生化和结构生物学分析

• 批准号: 42267534
• 负责人: Professor Dr. Matthias Geyer
• 金额: --
• 依托单位: Institut für Strukturbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2007
• 资助国家: 德国
• 起止时间: 2006-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/42267534?language=en
• 关键词: Biochemische; und; strukturbiologische; Analyse; der

The transition from transcription initiation to productive elongation in eukaryotic cells is highly regulated by the phosphorylation status of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). After transcription initiation, RNAPII pauses approximately 50-150 nucleotides downstream of the transcription start site. Release from this block requires the positive transcription elongation factor P-TEFb, which is a heterodimer composed of the cyclin-dependent kinase Cdk9 and the regulatory subunit Cyclin T. P-TEFb phosphorylates the repetitive hepta-repeat structure YSPTSPS of the CTD. Genome-wide studies suggest that the majority of eukaryotic genes are under the control of such promoter-proximal pausing. Misregulation of transcriptional elongation is involved in cancer, leukemia, myocardial hypertrophy and progression of HIV-1 infection to AIDS. We aim at analyzing the molecular and structural mechanisms that determine the activity and regulation of this transcription-controlling kinase. This research project focuses on the regulation of P-TEFb by activating factors such as Brd4 and the interaction and modification of P-TEFb substrates. In previous unpublished experiments we have determined that the C-terminal domain of Brd4 is able to activate P-TEFb and stimulate its catalytic activity over basal levels. We now want to analyze the specificity of substrate phosphorylation by the P-TEFb/Brd4 complex. Furthermore, we identified sequence homologies between Brd4 and HIV-1 Tat proteins, which shall by characterized by mutational studies. P-TEFb inhibition is mediated by the large 7SK snRNP complex composed of 7SK snRNA, Larp7, the coupling factor Hexim1 and P-TEFb. The spliceosomal SR protein SRSF2 has recently been identified as an additional subunit of 7SK snRNP, required for P-TEFb recruitment and activation. We want to generate this hexameric ribonucleoprotein complex including SRSF2 from recombinant protein expression for functional studies. Finally, the CDK/cyclin kinase pair CdkF1/CycH shall be analyzed for its ability to phosphorylate the CTD at Ser7 positions. Such phosphorylation mark is associated with transcription initiation and supposed to stimulate its recognition by P-TEFb. We expect that this research program will help understanding the regulation and substrate interaction of P-TEFb on a molecular level.

真核细胞中RNA聚合酶II (RNAPII) c端结构域（CTD）的磷酸化状态高度调控了转录起始到生产延伸的转变。转录起始后，RNAPII暂停在转录起始位点下游约50-150个核苷酸处。从这个阻滞区释放需要正转录延伸因子P-TEFb，它是由周期蛋白依赖性激酶Cdk9和调节亚基Cyclin t组成的异源二聚体。P-TEFb磷酸化CTD的重复七重重复结构YSPTSPS。全基因组研究表明，大多数真核生物基因都受到这种启动子-近端暂停的控制。转录延伸的错误调控涉及癌症、白血病、心肌肥大和HIV-1感染到艾滋病的进展。我们的目的是分析决定这种转录控制激酶的活性和调控的分子和结构机制。本课题主要研究Brd4等激活因子对P-TEFb的调控以及P-TEFb底物的相互作用和修饰。在之前未发表的实验中，我们已经确定Brd4的c端结构域能够激活P-TEFb并在基础水平上刺激其催化活性。我们现在想分析P-TEFb/Brd4复合物磷酸化底物的特异性。此外，我们确定了Brd4和HIV-1 Tat蛋白之间的序列同源性，这将通过突变研究来表征。P-TEFb抑制是由7SK snRNA、Larp7、偶联因子Hexim1和P-TEFb组成的大7SK snRNP复合物介导的。剪接体SR蛋白SRSF2最近被鉴定为7SK snRNP的另一个亚基，是P-TEFb募集和激活所必需的。我们希望从重组蛋白表达中生成包括SRSF2在内的六聚体核糖核蛋白复合物，用于功能研究。最后，CDK/cyclin激酶对CdkF1/CycH将分析其磷酸化CTD Ser7位点的能力。这种磷酸化标记与转录起始有关，并可能刺激P-TEFb对其的识别。我们期望这个研究项目将有助于在分子水平上理解P-TEFb的调控和底物相互作用。