Structural studies of eukaryotic transcription

申请人提供的描述：Mediator是一个在真核生物中保守的大的(25个多肽，Mw~1的丙二醛)多蛋白复合体，是在转录启动过程中负责向RNA聚合酶II(RNAPII)传递调节信号的关键辅助激活因子。介体基本上没有酶活性，人们普遍认为其作用机制必须基于分子相互作用和构象重排的调节。因此，对Mediator及其与RNAPII的相互作用的结构性理解对于阐明Mediator在启动过程中的作用最终如何影响细胞分化、发育和动态平衡是至关重要的。大分子电子显微镜(EM)是表征大的、动态的大分子组件的首选技术，因为它特别适合于提供关于它们的结构、构象变化和相互作用的信息。由这笔赠款支持的对介体的EM研究已经为实现对复合体的分子理解奠定了基础，在这里我们描述了对酵母和人类介体的进一步的EM、生化和功能分析，这些分析建立在我们以前工作的基础上，将提供对介体的结构、亚基组织和调节的详细了解，并揭示真核生物中介体的调节机制。在目标1中，我们将进行亚纳米分辨率的酵母介体(YMED)的EM分析，并研究对yMED组装、构象可变性和功能至关重要的亚基界面。在目标2中，我们将研究不同的因素如何控制介体的构象和相互作用来调节RNAPII的参与和转录启动，并获得关于介体-RNAPII全酶的详细结构信息。在目标3中，我们将利用从yMED的EM分析中获得的经验，对人类介体(HMED)的结构、亚单位组织和相互作用进行EM研究。对酵母和人类介体的平行分析将揭示与真核生物介体结构保守相关的调控机制的基本方面，同时突出更复杂的人类复合体特有的调控方面。我们的结果将提供对Mediator启动转录调控方式的分子理解，并帮助我们理解基因表达异常与肿瘤发生之间的直接联系。我们提出的工作将继续我们将结构分析与生化和功能研究相结合的战略，并将关键取决于1)与Pawel Penczek(UT Houston)小组合作开发的新型EM图像分析方法的应用，该小组专门为研究动态复合体的结构而量身定做；2)与Joan和Ron Conaway(Stowers Institute)领导的小组进行强有力的持续合作，他们是人类介体生化和功能研究的领导者。

DESCRIPTION (provided by applicant): Mediator, a large (25 polypeptides, MW ~1 MDa) multi-protein complex conserved throughout eukaryotes is the key coactivator responsible for conveying regulatory signals to RNA polymerase II (RNAPII) during transcription initiation. Mediator has essentially no enzymatic activity and it is generally agreed that its mechanism must be based on modulation of molecular interactions and conformational rearrangements. Therefore, a structural understanding of Mediator and its interaction with RNAPII is essential to elucidate how Mediator's role during initiation ultimately influences cell differentiation, development, and homeostasis. Macromolecular electron microscopy (EM) is the technique of choice for characterization of large, dynamic macromolecular assemblies because it is uniquely suited to provide information about their structure, conformational changes, and interactions. EM studies of Mediator supported by this grant have set the stage to achieve a molecular understanding of the complex and here we describe further EM, biochemical, and functional analyses of yeast and human Mediators that build on our previous work and will provide a detailed understanding of the structure, subunit organization, and regulation of Mediator conformation and interactions, and reveal the mechanism of regulation by Mediator across eukaryotes. In Aim 1 we will pursue EM analysis of yeast Mediator (yMED) at subnanometer resolution and investigate subunit interfaces critical for yMED assembly, conformational variability, and function. In Aim 2 we will study how different factors control Mediator conformation and interactions to modulate RNAPII engagement and transcription initiation, and obtain detailed structural information about the Mediator-RNAPII holoenzyme. In Aim 3 we will leverage the experience gained from EM analysis of yMED to pursue EM studies of the structure, subunit organization, and interactions of human Mediator (hMED). Parallel analysis of yeast and human Mediators will reveal fundamental aspects of the regulation mechanism related to structural conservation of Mediator across eukaryotes, while highlight aspects of regulation specific to the more intricate human complex. Our results will provide a molecular understanding of the way in which Mediator enables regulation of transcription initiation, and help us understand the direct connection between dysregulation of gene expression and oncogenesis. The work we propose continues our strategy of combining structural analysis with biochemical and functional studies, and will depend critically on 1) application of novel EM image analysis approaches developed in collaboration with the group of Pawel Penczek (UT Houston) specifically tailored to study the structure of dynamic complexes; 2) a strong, ongoing collaboration with the group led by Joan and Ron Conaway (Stowers Institute), leaders in biochemical and functional studies of human Mediator.