Structural studies of eukaryotic transcription

摘要 细胞的分化、发育和动态平衡依赖于基因表达的调节，这是 主要集中在DNA转录起始过程。在转录启动过程中，一个大的 真核生物中保守的多蛋白复合体向RNA聚合酶II传递调控信号 (RNAPII)，负责转录所有蛋白质编码基因的酶。取决于具体情况 生物体，介体可以包括25-29种不同的多肽(总分子量1-1.5MDa)，组织成Head，Medium， Tail和CDK8Kinase(CKM)模块，但其酶活性仅限于单一的CDK8激酶亚基。 越来越多的证据表明，一种在很大程度上基于构象重排的机制 与RNAPII的介体相互作用。因此，对调解人的结构和其 构象动力学对于阐明该复合体如何调节起始是至关重要的。 大分子电子显微镜(CRYO-EM)是表征大型、 动态大分子组装。在过去的几年里，对介体的低温电磁研究 极大地提高了我们对该复合体的分子理解。在这里，我们建议低温EM，生化， 以及对酵母和哺乳动物媒介的功能研究，这些研究建立在我们之前的工作基础上，这将揭示 在分子细节中，关键因子调节介体构象和相互作用的方式 RNAPII，导致转录启动的调节。 在目标1中，我们将计算小鼠(MmMED)和人类(HsMED)介体在近原子状态下的低温EM图 解析、本地化后生动物特定的亚基，并确定这些亚基如何影响介体结构 重排和RNAPII相互作用。这些结果将提供对原子分辨率的理解 哺乳动物的中介结构和揭示转录特定细节的结构基础 哺乳动物介体的调节。 在目标2中，我们将使用冷冻-EM和生物化学来确定CKM结合对介体的影响 构象和RNAPII结合，并将研究CKM与介体相互作用的调节。这将是 了解中介如何与CKM交互以及随之而来的结构变化 影响与RNAPII、全酶形成和最终转录的介体关联 入会仪式。 在目标3中，我们将使用冷冻-EM、图像分析和生物化学来了解激活剂和 抑制子对酵母和哺乳动物介体的影响影响介体构象，与RNAPII和 基因表达。这些研究将揭示与激活物和抑制物的相互作用，其中 通常，靶子单元(主要在Tail模块中)不直接参与RNAPII相互作用，可以 最终影响介体对转录启动的调控。 我们提出的研究结果将提供对哺乳动物介体结构的详细了解， 并揭示了构象调控和相互作用如何在整体上实现中介的转录调控 真核生物。对酵母和哺乳动物介体的平行分析将揭示 与真核细胞中介体结构保守相关的调控机制，同时强调 特定于更复杂的哺乳动物介体的调控方面。我们提议的工作继续我们的 将结构分析与生化和功能研究相结合的战略，并将严重依赖于 1)应用最先进的低温EM，我的团队在这方面有相当的专业知识；2)强大、持续 与哺乳动物生化和功能研究领域的领先者研究小组合作 调解人。

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.