The Cdk8 kinase module regulates Mediator-RNA polymerase II interaction

We use biochemically reconstituted transcription initiation components including the Mediator core and Cdk8 kinase modules (CKM) to investigate the function of the CKM in regulating the Mediator-RNA polymerase II interaction in a highly purified system. We use cross-linking coupled to mass spectrometry to map the interaction of the CKM and core Mediator, and in vitro phosphorylation assays followed by phosphopeptide enrichment coupled to mass spectrometry to identify Cdk8 phosphorylation targets in this context and biochemically dissect their functions. Finally, we investigate the function of phosphorylation by Cdk8 on transcription in vivo. Based on that, we propose a model to integrate the phosphorylation-dependent and -independent functions of the CKM in transcription initiation.