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

点击翻译按钮获取中文摘要

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 In previous unpublished experiments we have determined that the C-terminal domain of Brd4 is able to activate P-TEFb and stimulate its catalytic activity over basal levels.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