The functional and molecular architecture of the CAK complex

通用转录因子II H(TFIIH)在依赖RNA聚合酶II(RNA Pol II)的转录和核苷酸切除修复(NER)中起着核心作用。TFIIH总共由10个亚基组成，其中Xpb、Xpd、p62、p52、p44、p34和p8组装成其核心。CDK7、MAT1和Cyclin H亚基组成了细胞周期蛋白激活的激酶(CAK)模块，这是一个在启动子清除、近端停顿、共转录染色质修饰和终止中起关键作用的激酶复合体，因此对基于RNA Pol II的转录是必不可少的。在过去的几年里，在转录和核苷酸切除修复(NER)的背景下，TFIIH的几个冷冻EM结构已经被解决。然而，尽管这些结构提供了对TFIIH核心的巨大洞察，但到目前为止还没有描述CAK以及最重要的CDK7活性形式的结构信息。我们之前已经证明，真核模式生物嗜热毛壳菌的所有核心TFIIH成分都可以生产出足够数量和质量的TFIIH，用于生化和结构分析。此外，在原理分析的证明中，我们证明了从嗜热梭菌获得的XPD数据可以与从人类XPD获得的数据直接相关。我们现在已经将这种方法扩展到真菌CAK(CtCAK)的三个亚基，并成功地确定了包含所有三种蛋白质的复合体的初始结构。基于这些数据，我们的目标是获得包含结合核苷酸以及模拟RNA POLII亚基Rpb1 C-末端尾部的多肽的复合体。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突变。我们的研究还将包括对核心TFIIH上的活性CAK复合体的分析。即在TFIIH亚基内可能发生的磷酸化事件，这可能在核苷酸切除修复(NER)或转录中具有调节功能。此外，我们将把我们的分析扩展到人类CAK，我们也可以将其纯化为同质性。根据我们的功能突变和ctCAK的原位磷酸化数据，我们将讨论对CAK复合体的形成和活性至关重要的特定残基，并分析它们对转录和修复的影响。综上所述，这些研究将为CDK7与MAT1和Cyclin H的复合体如何变得高度特异于Rbp1的C末端提供重要的新见解，并可能在转录或修复中承担对TFIIH的调节功能。重要的是，CDK7也被证明是一个很有前途的肿瘤治疗靶点，THZ1及其继任者等抑制剂显示出高度的抗增殖活性。因此，对CAK复合体中CDK7活性形式的洞察应有助于开发新的有效抑制剂。

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.