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

在真核细胞中，从转录起始到生产性延伸的转变受到RNA聚合酶II（RNAPII）的C-末端结构域（CTD）的磷酸化状态的高度调节。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。从该阻断中释放需要正转录延伸因子P-TEFb，其是由细胞周期蛋白依赖性激酶Cdk 9和调节亚基细胞周期蛋白T组成的异源二聚体。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。转录延长的失调与癌症、白血病、心肌肥大和HIV-1感染向AIDS的进展有关。我们的目的是分析的分子和结构的机制，确定这种转录控制激酶的活性和调节。本研究项目的重点是Brd 4等活化因子对P-TEFb的调控以及P-TEFb底物的相互作用和修饰。在先前未发表的实验中，我们已经确定Brd 4的C-末端结构域能够激活P-TEFb并刺激其催化活性超过基础水平。我们现在想分析P-TEFb/Brd 4复合物对底物磷酸化的特异性。此外，我们确定了Brd 4和HIV-1达特蛋白之间的序列同源性，这将通过突变研究来表征。P-TEFb抑制由7SK snRNA、Larp 7、偶联因子Hexim 1和P-TEFb组成的大7SK snRNP复合物介导。剪接体SR蛋白SRSF 2最近被鉴定为7SK snRNP的额外亚基，其为P-TEFb募集和活化所需。我们希望从重组蛋白表达中产生包括SRSF 2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CdkF 1/CycH在Ser 7位置磷酸化CTD的能力。这种磷酸化标记与转录起始相关，并被认为刺激其被P-TEFb识别。我们希望这项研究计划将有助于在分子水平上了解P-TEFb的调控和底物相互作用。

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 In previous unpublished experiments we have determined that the C-terminal domain of Brd4 is able to activate P-TEFb and stimulate its catalytic activity over basal levels.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