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 In previous unpublished experiments we have determined that the C-terminal domain of Brd4 is able to activate P-TEFb and stimulate its catalytic activity over basal levels.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.

真核细胞中从转录起始到有效延伸的转变受到 RNA 聚合酶 II (RNAPII) C 末端结构域 (CTD) 磷酸化状态的高度调控。转录起始后，RNAPII 在转录起始位点下游约 50-150 个核苷酸处暂停。该块的释放需要正转录延伸因子 P-TEFb，它是由细胞周期蛋白依赖性激酶 Cdk9 和调节亚基 Cyclin T 组成的异二聚体。P-TEFb 磷酸化 CTD 的重复七重复结构 YSPTSPS。全基因组研究表明，大多数真核基因都受到这种启动子近端暂停的控制。转录延伸的失调与癌症、白血病、心肌肥大以及 HIV-1 感染进展为艾滋病有关。我们的目标是分析决定这种转录控制激酶的活性和调节的分子和结构机制。该研究项目的重点是通过激活Brd4等因子来调节P-TEFb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 并刺激其催化活性超过基础水平。我们现在想要分析 P-TEFb/Brd4 复合物对底物磷酸化的特异性。此外，我们还鉴定了 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、耦合因子 Hexim1 和 P-TEFb 组成的大型 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 募集和激活所需的。我们希望通过重组蛋白表达生成包括 SRSF2 的六聚体核糖核蛋白复合物，用于功能研究。最后，应分析 CDK/细胞周期蛋白激酶对 CdkF1/CycH 磷酸化 CTD Ser7 位点的能力。这种磷酸化标记与转录起始相关，并被认为可以刺激 P-TEFb 对其进行识别。我们期望该研究计划将有助于在分子水平上了解 P-TEFb 的调节和底物相互作用。