The functional and molecular architecture of the CAK complex

CAK复合物的功能和分子结构

• 批准号: 448287206
• 负责人: Professorin Dr. Caroline Kisker
• 金额: --
• 依托单位: Rudolf-Virchow-Zentrum - Center for Integrative and Translational Bioimaging
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/448287206?language=en
• 关键词: functional; molecular; architecture; CAK; complex

The general transcription factor II H (TFIIH) plays a central role in both RNA Polymerase II (RNA Pol II) dependent transcription and nucleotide excision repair (NER). In its entirety TFIIH consists of a total of 10 subunits of which XPB, XPD, p62, p52, p44, p34, and p8 assembles into its core. The CDK7, MAT1, and CyclinH subunits constitute the cyclin-activated kinase (CAK) module, a kinase complex that is crucially involved in promoter clearance, proximal pausing, co-transcriptional chromatin modification, and termination and thus essential to RNA Pol II based transcription. Several cryo-EM structures of TFIIH in the context of transcription and nucleotide excision repair (NER) have been solved in the last couple of years. However, while these structures provide tremendous insights into the TFIIH core, no structural information is available so far that describes the CAK and, most importantly, the active form of CDK7. We have previously shown that all core TFIIH components from the eukaryotic model organism Chaetomium thermophilum can be produced in sufficient quantity and quality for biochemical and structural analyses. Furthermore, in a proof of principle analysis we demonstrated that data obtained for XPD from C. thermophilum can be directly correlated with those from human XPD. We have now extended this approach towards the three fungal CAK (ctCAK) subunits and successfully determined an initial structure of a complex containing all three proteins. Based on these data we aim to obtain complexes containing bound nucleotides as well as peptides mimicking the C-terminal tail of the RNA Pol II subunit Rpb1. The structural analysis will be accompanied by biochemical studies with an emphasis on enzyme kinetics and structure based functional mutagenesis. Our studies will also include the analysis of an active CAK complex on core TFIIH. i.e. possible phosphorylation events within TFIIH subunits which may have a regulatory function in nucleotide excision repair (NER) or transcription. In addition, we will extend our analyses to human CAK, which we can also purify to homogeneity. Based on our functional mutagenesis and in situ phosphorylation data of ctCAK, we will address specific residues which are crucial for CAK complex formation and activity, and analyze their effects on transcription and repair. Combined, these studies will provide important new insights into how CDK7 in complex with MAT1 and Cyclin H becomes highly specific towards the C-terminus of Rbp1 and may assume a regulatory function towards TFIIH in transcription or repair. Importantly, CDK7 was also shown to be a promising target for tumor therapy and inhibitors such as THZ1 and its successors display high anti-proliferative activity. Insights into the active form of CDK7 in the CAK complex should therefore aid in the development of new potent inhibitors.

一般转录因子II H（TFIIH）在RNA聚合酶II（RNA Pol II）依赖性转录和核苷酸切除修复（NER）中起核心作用。TFIIH整体由总共10个亚基组成，其中XPB、XPD、p62、p52、p44、p34和p8组装成其核心。CDK 7、MAT 1和细胞周期蛋白H亚基构成了细胞周期蛋白激活激酶（CAK）模块，这是一种激酶复合物，其在启动子清除、近端暂停、共转录染色质修饰和终止中起关键作用，因此对于基于RNA Pol II的转录至关重要。在转录和核苷酸切除修复（NER）的背景下，TFIIH的几个cryo-EM结构在过去几年中已经得到解决。然而，虽然这些结构提供了对TFIIH核心的巨大见解，但迄今为止还没有结构信息描述CAK，最重要的是，CDK 7的活性形式。 我们以前已经表明，所有核心TFIIH组件从真核模式生物嗜热毛壳菌可以产生足够的数量和质量的生化和结构分析。此外，在原理分析的证明中，我们证明了从C.嗜热菌的DNA序列可以与来自人XPD的DNA序列直接相关。我们现在已经将这种方法扩展到三种真菌CAK（ctCAK）亚基，并成功确定了包含所有三种蛋白质的复合物的初始结构。基于这些数据，我们的目标是获得复合物含有结合的核苷酸，以及模仿的RNA Pol II亚基Rpb 1的C-末端尾巴的肽。结构分析将伴随着生化研究，重点是酶动力学和基于结构的功能性诱变。我们的研究还将包括对核心TFIIH上的活性CAK复合物的分析。即TFIIH亚基内可能的磷酸化事件，其可能在核苷酸切除修复（NER）或转录中具有调节功能。此外，我们将我们的分析扩展到人类CAK，我们也可以纯化到同质。基于我们的ctCAK的功能突变和原位磷酸化数据，我们将解决特定的残基，CAK复合物的形成和活性是至关重要的，并分析其对转录和修复的影响。结合，这些研究将提供重要的新见解CDK 7与MAT 1和细胞周期蛋白H的复合物如何变得高度特异性对Rbp 1的C-末端，并可能承担对转录或修复TFIIH的调节功能。重要的是，CDK 7也被证明是肿瘤治疗的一个有前途的靶点，抑制剂如THZ 1及其后继者显示出高的抗增殖活性。因此，深入了解CAK复合物中CDK 7的活性形式有助于开发新的有效抑制剂。