Structural insights into the Argonaute-containing CDK8 complex

摘要 在真核生物中，转录调节因子包含一个可解离的CDK8激酶模块(CKM)，它调节基因 通过激酶依赖和非依赖功能表达。人的CKM由Med13组成， MED12、CDK8和CycC亚基，在神经发育障碍和 多发性癌症，如乳腺癌和结直肠癌。尽管CKM和人类之间存在广泛的联系 疾病，CKM在基因转录调控中的基本机制仍然知之甚少。 长期目标是阐明CKM调节基因转录的分子机制。 通过它的激酶功能和核酸相互作用。本申请的总体目标是(I) 揭示CKM亚基的结构基础和功能作用，(II)阐明CDK8的激活机制 以及它识别磷酸化底物的方式，以及(Iii)研究功能 Med13与核酸的相互作用。在这项申请中提出了三个目标。目标1是 揭示酵母CKM的高分辨率结构并了解CDK8是如何被MED12激活的 并结合衬底。我们将使用单粒子低温电磁来揭示 互动 多么 这个 目标 酵母菌CKM的整体结构及其特性 以CDK8为底物。我们将使用生物化学的方法来阐明 CDK8是由MED12通过一种非规范的机制激活的。在目标2中，我们将描述 用生化方法研究Med13的核酸结合特性并鉴定其结合 使用下一代测序技术。在目标3中，我们将使用我们对yCKM的详细知识来加强 我们的知识是从研究人类CKM的结构、底物结合和疾病突变中获得的。 拟议数 激活剂 调节 这个 研究具有重要意义，因为它们将为 CKM的功能和核酸相互作用揭示了CKM的基本机制 关于基因表达的。最终，我们的结果将提供 ， 发展的结构性基础 用于治疗CDK8驱动的人类疾病的激酶抑制剂。

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.