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

基本信息

项目摘要

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 In previous unpublished experiments we have determined that the C-terminal domain of Brd4 is able to activate P-TEFb and stimulate its catalytic activity over basal levels.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.
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。全基因组的研究表明,大多数真核基因的控制下,这种启动子近端暂停。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。

项目成果

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Professor Dr. Matthias Geyer其他文献

Professor Dr. Matthias Geyer的其他文献

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{{ truncateString('Professor Dr. Matthias Geyer', 18)}}的其他基金

Structural and functional analysis of transcription regulating kinases Cdk10 and Cdk11
转录调节激酶 Cdk10 和 Cdk11 的结构和功能分析
  • 批准号:
    226824387
  • 财政年份:
    2012
  • 资助金额:
    --
  • 项目类别:
    Research Grants
Structure-function analysis of mammalian formins FMNL1 and FMNL2
哺乳动物formins FMNL1和FMNL2的结构-功能分析
  • 批准号:
    170435620
  • 财政年份:
    2010
  • 资助金额:
    --
  • 项目类别:
    Priority Programmes
Analyse der Komplexbildung und Membranassoziation der heterotetramen Adaptorproteine AP -1 und AP-2 mit Cargoproteinen HIV-1 Nef und CD4
异四联接头蛋白 AP -1 和 AP-2 与货物蛋白 HIV-1 Nef 和 CD4 的复合物形成和膜关联分析
  • 批准号:
    93860933
  • 财政年份:
    2008
  • 资助金额:
    --
  • 项目类别:
    Research Grants
Biochemische Charakterisierung und Struktur-Funktionsbeziehung der GTP-bindenden Domäne des Trans-Aktivatorproteins CIITA
反式激活蛋白CIITA GTP结合域的生化特征及结构功能关系
  • 批准号:
    5405025
  • 财政年份:
    2003
  • 资助金额:
    --
  • 项目类别:
    Research Grants
Design und Charakterisierung eines Moleküls zur Inhibierung des HIV Pathogenesefaktors Nef
抑制 HIV 发病因子 Nef 的分子的设计和表征
  • 批准号:
    5395763
  • 财政年份:
    2003
  • 资助金额:
    --
  • 项目类别:
    Research Grants
Biochemical and structural analysis of the NLRP3 inflammasome
NLRP3炎症小体的生化和结构分析
  • 批准号:
    504829198
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
    Research Grants

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