The functional and molecular architecture of the CAK complex

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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.