Mechanisms of Transcriptional Control by the Mediator Complex Subunit Cdk8

Abstract

Transcription is a tightly regulated process wherein RNA polymerases (RNAPs) use DNA as a template to synthesize RNA. In eukaryotes, RNAPII transcribes all protein-coding and many types of non-protein-coding genes. The multi-subunit Mediator complex functions in RNAPII transcription regulation by acting as a bridge between RNAPII and transcription factors. Cyclin-dependent kinase 8 (Cdk8) is the only enzymatic subunit in the entire Mediator complex, and it resides in a dissociable module with CycC, Med12, and Med13. Cdk8 regulates transcription by phosphorylating transcription factors in response to environmental signals. However, the full extent of all Cdk8 substrates and the mechanisms by which Cdk8 itself is regulated are not completely understood. As a cyclin-dependent kinase, partial Cdk8 activation in vitro requires interaction with CycC, its cyclin partner. Full activation is achieved by the stabilization of the Cdk8 T-loop (a region important for catalysis) by the Med12 N-terminus, but whether this interaction regulates Cdk8 activity in vivo is not known. My project examined the significance of the Cdk8-Med12 interaction in vivo, revealing that this interaction is necessary for normal Cdk8 function. Furthermore, my work showed that this interaction is dynamic and condition-specific, suggesting a means to regulate Cdk8 activity in response to environmental signals in vivo. My thesis also advanced work aimed at identifying novel Cdk8 substrates by cloning, expressing, and purifying putative substrates for in vitro Cdk8 kinase analysis. Understanding Cdk8 function and the factors that regulate its activity is important for understanding how organisms fine-tune transcription in response to environmental signals. Furthermore, given that Cdk8 is frequently amplified or overexpressed in cancer, my work provides insight that may be relevant to our understanding of this disease state.