Structural studies of eukaryotic transcription

真核转录的结构研究

• 批准号: 10004054
• 负责人: Francisco J Asturias
• 金额: $ 47.27万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10004054
• 关键词: Affect; Binding; Biochemical; Biochemistry; Code; Collaborations; Complex; Cryoelectron Microscopy; DNA; Development; Disease; Electron Microscopy; Enzymes; Eukaryota; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Head; Holoenzymes; Homeostasis; Human; Image Analysis; Malignant Neoplasms; Maps; Mediator of activation protein; Molecular; Molecular Conformation; Multiprotein Complexes; Mus; Organism; Phosphotransferases; Play; Process; Proteins; Public Health; RNA Polymerase II; Regulation; Research; Resolution; Role; Signal Transduction; Structure; Tail; Techniques; Transcription Initiation; Transcriptional Regulation; Work; Yeasts; base; developmental disease; experimental study; macromolecular assembly; polypeptide; public health relevance

ABSTRACT Cellular differentiation, development and homeostasis depend on regulation of gene expression, which is largely focused on the DNA transcription initiation process. During transcription initiation, Mediator, a large multi-protein complex conserved throughout eukaryotes, conveys regulatory signals to RNA polymerase II (RNAPII), the enzyme responsible for transcription of all protein-coding genes. Depending on the specific organism, Mediator can include 25-29 different polypeptides (total MW 1-1.5MDa) organized into Head, Middle, Tail and CDK8 Kinase (CKM) modules, but its enzymatic activity is limited to a single Cdk8 kinase subunit. Mounting evidence points to a mechanism largely based on conformational rearrangements that modulate Mediator interaction with RNAPII. Consequently, a detailed understanding of Mediator structure and its conformational dynamics is essential to elucidate how the complex regulates initiation. Macromolecular electron microscopy (cryo-EM) is the technique of choice for characterization of large, dynamic macromolecular assemblies. In the last couple of years, cryo-EM studies of Mediator have dramatically advanced our molecular understanding of the complex. Here we propose cryo-EM, biochemical, and functional studies of yeast and mammalian Mediators that build on our previous work, and that will reveal in molecular detail the way in which critical factors modulate Mediator conformation and interaction with RNAPII, bringing about regulation of transcription initiation. In Aim 1 We will calculate cryo EM maps of mouse (MmMED) and human (HsMED) Mediators at near-atomic resolution, localize metazoan-specific subunits, and determine how these subunits affect Mediator structural rearrangements and RNAPII interaction. These results will provide an atomic-resolution understanding of mammalian Mediator structure and reveal the structural basis for specific details of transcription regulation by mammalian Mediator. In Aim 2 we will use cryo-EM and biochemistry to determine the effect of CKM binding on Mediator conformation and RNAPII association, and will investigate regulation of CKM interaction with Mediator. This will lead to an understanding of how Mediator interaction with the CKM and concomitant structural changes influence Mediator association with RNAPII, holoenzyme formation and, ultimately, transcription initiation. In Aim 3 we will use cryo-EM, image analysis and biochemistry to understand how binding of activators and repressors to yeast and mammalian Mediators influences Mediator conformation, interaction with RNAPII and gene expression. These studies will reveal how interaction with activators and repressors, which generally target subunits (mostly in the Tail module) not directly involved in RNAPII interaction, can ultimately have an effect on regulation of transcription initiation by Mediator. Results from the studies we propose will provide a detailed understanding of mammalian Mediator structure, and reveal how conformational regulation and interactions enable transcription regulation by Mediator in all eukaryotes. Parallel analysis of yeast and mammalian Mediators will reveal fundamental aspects of the regulation mechanism related to structural conservation of Mediator across eukaryotes, while highlighting aspects of regulation specific to the more intricate mammalian Mediator. The work we propose continues our strategy of combining structural analysis with biochemical and functional studies, and will depend critically on 1) application of state-of-the-art cryo-EM, in which my group has considerable expertise; 2) strong, ongoing collaborations with research groups that are leaders in biochemical and functional studies of mammalian Mediator.

摘要 细胞分化、发育和稳态依赖于基因表达的调节， 主要集中在DNA转录起始过程。在转录起始期间，介体，一个大的 一种在真核生物中保守的多蛋白复合物，将调节信号传递给RNA聚合酶II RNAPII是负责所有蛋白质编码基因转录的酶。根据具体 介体可以包括25-29种不同的多肽（总MW 1-1.5MDa），其被组织成头部，中部， 尾和CDK 8激酶（CKM）模块，但其酶活性仅限于单个Cdk 8激酶亚基。 越来越多的证据表明，在很大程度上基于构象重排的机制， 介体与RNAPII的相互作用。因此，详细了解Mediator结构及其 构象动力学对于阐明复合物如何调节起始是必不可少的。 大分子电子显微镜（cryo-EM）是表征大， 动态大分子组装体在过去的几年里，Mediator的冷冻电镜研究 极大地推进了我们对复合体的分子理解。在这里，我们提出冷冻EM，生化， 酵母和哺乳动物介体的功能研究建立在我们以前的工作，这将揭示 在分子细节中，关键因素调节介体构象和与 RNAPII，引起转录起始的调节。 在目标1中，我们将计算小鼠（MmMED）和人（HsMED）介体在近原子水平的冷冻EM图。 分辨率，定位后生动物特异性亚基，并确定这些亚基如何影响介体结构 重组和RNAPII相互作用。这些结果将提供一个原子分辨率的理解， 哺乳动物介体结构，并揭示了转录的具体细节的结构基础 哺乳动物介体的调节。 在目标2中，我们将使用冷冻电镜和生物化学来确定CKM结合对介体的影响。 构象和RNAPII关联，并将研究CKM与Mediator相互作用的调节。这将 了解Mediator如何与CKM相互作用以及伴随的结构变化 影响介体与RNAPII、全酶形成和最终转录的关联 初始化。 在目标3中，我们将使用冷冻电镜，图像分析和生物化学来了解激活剂和 酵母和哺乳动物介体的阻遏物影响介体构象，与RNAPII的相互作用， 基因表达。这些研究将揭示如何相互作用的激活剂和阻遏物， 通常靶向不直接参与RNAPII相互作用的亚基（主要在Tail模块中）， 最终通过介体对转录起始的调节产生影响。 我们提出的研究结果将提供对哺乳动物中介体结构的详细了解， 并揭示了构象调控和相互作用如何使介体在所有的转录调控 真核生物酵母和哺乳动物介体的平行分析将揭示 与真核生物中介体结构保守相关的调节机制，同时强调 更复杂的哺乳动物中介体的特定调节方面。我们建议的工作继续我们的 将结构分析与生物化学和功能研究相结合的战略，并将严重依赖于 1)应用最先进的冷冻EM，我的团队在这方面具有相当的专业知识; 2）强大，持续 与哺乳动物生物化学和功能研究的领导者研究小组合作 调解员