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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

基本信息

批准号：
226824387
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：
2012
资助国家：
德国
起止时间：
2011-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/226824387?language=en
关键词：
Structural functional analysis transcription regulating

项目摘要

The regulation of transcription is essential for gene expression in eukaryotic cells. Different sets of phosphorylations in the C-terminal domain of the largest subunit of RNA polymerase II are thought to correlate with the various phases of transcription initiation, promoter-proximal pausing, elongation, and termination. The phosphorylations are set in part by cyclin-dependent kinases (CDKs), whose timed interplay orchestrates the transcription cycle. In the first funding period we have determined the crystal structures of Cdk12/CycK and Cdk13/CycK and analysed in collaboration the inhibition of the kinase activity by small molecular compounds dinaciclib and THZ531. In the second funding period we aim at analysing the complexes Cdk10/cyclin M and Cdk11/cyclin L, which are thought to regulate transcription and RNA splicing. In preliminary work we have purified the Cdk10/CycM complex from baculo-virus infected insect cells to homogeneity, analysed its kinase activity, and performed initial crystallization trials. In future experiments we want to determine the Cdk10/CycM structure, characterize the phosphorylation activity and specificity, analyse the substrate profile by an analogue-sensitive mutant kinase, generate Cdk10 and Cdk11 deficient cell lines by CRISPR/Cas, perform a structure-function analysis of Cdk11/CycL and finally determine the crystal structures of Cdk12/CycK or Cdk13/CycK from the first funding period bound to molecular inhibitors. These studies will significantly contribute to our understanding of the molecular mechanism of transcription regulation.

转录调控是真核细胞基因表达所必需的。RNA聚合酶II的最大亚基的C-末端结构域中的不同磷酸化被认为与转录起始、启动子近端暂停、延伸和终止的各个阶段相关。磷酸化部分由细胞周期蛋白依赖性激酶（CDK）设置，其定时相互作用协调转录周期。在第一个资助期内，我们确定了Cdk 12/CycK和Cdk 13/CycK的晶体结构，并合作分析了小分子化合物dinaciclib和THZ 531对激酶活性的抑制作用。在第二个资助期，我们的目标是分析复合物Cdk 10/细胞周期蛋白M和Cdk 11/细胞周期蛋白L，这被认为是调节转录和RNA剪接。在初步工作中，我们已经纯化的Cdk 10/CycM复合物从杆状病毒感染的昆虫细胞的同质性，分析其激酶活性，并进行初步的结晶试验。在未来的实验中，我们希望确定Cdk 10/CycM结构，表征磷酸化活性和特异性，通过类似物敏感的突变激酶分析底物谱，通过CRISPR/Cas产生Cdk 10和Cdk 11缺陷细胞系，对Cdk 11/CycL进行结构-功能分析，最终确定Cdk 12/CycK或Cdk 13/CycL的晶体结构。第一个资助期的CycK与分子抑制剂结合。这些研究将有助于我们理解转录调控的分子机制。