Structural insights into the Argonaute-containing CDK8 complex

摘要 在真核生物中，转录介体包含一个可分离的Cdk 8激酶模块（CKM）， 通过激酶依赖性和非依赖性功能表达。人CKM由Med 13组成， Med 12、Cdk 8和CycC亚基，它们在神经发育障碍中突变或扩增， 多种癌症，如乳腺癌和结肠直肠癌。尽管CKM和人类之间存在广泛的联系， 尽管CKM在疾病中的作用机制尚不清楚，但CKM在基因转录调控中的基本机制仍知之甚少。 长期目标是阐明CKM调节基因转录的分子机制 通过其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Cdk 8的激活机制 以及它识别磷酸化底物的方式，以及（iii）研究功能性 Med 13与核酸之间的相互作用。在本申请中提出了三个目的。目标1： 揭示酵母CKM的高分辨率结构，并了解Cdk 8如何被Med 12激活 并结合底物。我们将使用单粒子低温电镜来揭示 相互作用 如何 的 目标 酵母CKM整体结构及其 Cdk 8基板我们将使用生物化学的方法来阐明详细的机制， Cdk 8激酶通过非经典机制被Med 12激活。在目标2中，我们将描述 使用生化方法研究Med 13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yCKM的详细知识来加强 我们的知识来自对人类CKM结构、底物结合和疾病突变的研究。 提出 激酶 调控 的 研究是重要的，因为它们将提供有价值的结构和功能的见解， CKM的功能和核酸相互作用，揭示CKM在 的基因表达。最终，我们的结果将提供 , 发展的结构基础 激酶抑制剂来治疗Cdk 8驱动的人类疾病。

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.