Structural and functional analysis of transcription regulating kinases Cdk10 and Cdk11

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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.