Structural insights into the Argonaute-containing CDK8 complex

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Abstract In eukaryotes, transcription Mediator contains a dissociable Cdk8 kinase module (CKM) that regulates gene expression through kinase-dependent and -independent functions. The human CKM consists of Med13, Med12, Cdk8, and CycC subunits, which are either mutated or amplified in neurodevelopmental disorders and multiple cancers, such as breast and colorectal cancers. Despite extensive links between CKM and human diseases, the fundamental mechanisms of CKM in regulation of gene transcription remain poorly understood. The long-term goal is to elucidate the molecular mechanisms by which CKM regulates gene transcription through its kinase function and nucleic acid interactions. The overall objectives in this application are to (i) reveal the structure basis and functional roles of CKM subunits, (ii) elucidate the activation mechanism of Cdk8 and the way in which it recognizes substrates for phosphorylation, and (iii) investigate the functional interactions between Med13 and nucleic acids. Three aims are proposed in this application. Aim 1 is to uncover the high-resolution structure of yeast CKM and understand how Cdk8 becomes activated by Med12 and binds substrates. We will use single-particle cryo-EM to reveal interactions how the targets the overall structure of yeast CKM and its with Cdk8 substrates. We will use biochemical approaches to elucidate the detailed mechanism of Cdk8 kinase is activated by Med12 through a non-canonical mechanism. In Aim 2 , we will characterize nucleic acid binding properties of Med13 using biochemical approaches as well as identify its binding using next-generation sequencing. In Aim 3, we willuse our detailed knowledge of yCKM to reinforce our knowledge gained from studies of human CKM's structure, substrate binding, and disease mutations. proposed kinase regulation The studies are significant because they will provide valuable structural and functional insights into the function and nucleic acids interaction of CKM revealing the fundamental mechanisms of CKM in of gene expression. Ultimately, our results will offer , a structural foundation for development of kinase inhibitors to treat Cdk8-driven human diseases.