Structural studies of eukaryotic transcription

描述（由申请人提供）：中介体，一个大的（25多肽，MW ~ 1mda）多蛋白复合物，在整个真核生物中保守，是转录起始过程中负责向RNA聚合酶II （RNAPII）传递调控信号的关键辅激活因子。介体基本上没有酶活性，普遍认为其机制必须基于分子相互作用和构象重排的调节。因此，了解Mediator的结构及其与RNAPII的相互作用对于阐明Mediator在起始过程中最终如何影响细胞分化、发育和稳态至关重要。大分子电子显微镜（EM）是表征大型动态大分子组件的首选技术，因为它非常适合提供有关其结构，构象变化和相互作用的信息。由该基金支持的Mediator的EM研究为实现对该复合体的分子理解奠定了基础，在这里，我们将在我们之前的工作基础上进一步描述酵母和人类Mediator的EM、生化和功能分析，并将提供对Mediator构象和相互作用的结构、亚基组织和调节的详细了解，并揭示Mediator在真核生物中的调节机制。在目标1中，我们将在亚纳米分辨率下对酵母介质（yMED）进行EM分析，并研究对yMED组装、构象变变性和功能至关重要的亚基界面。在Aim 2中，我们将研究不同的因素如何控制Mediator的构象和相互作用来调节RNAPII的接合和转录起始，并获得关于Mediator-RNAPII全酶的详细结构信息。在目标3中，我们将利用从hMED的EM分析中获得的经验来进行人类中介体（hMED）的结构、亚基组织和相互作用的EM研究。酵母和人类介质的平行分析将揭示与真核生物中介质结构保护相关的调节机制的基本方面，同时突出更复杂的人类复合物的特定调节方面。我们的研究结果将提供一个分子理解的方式，调解使转录起始调节，并帮助我们了解基因表达失调和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策略，并将主要依赖于1)与Pawel Penczek （UT Houston）团队合作开发的新型EM图像分析方法的应用，该方法专门用于研究动态复合物的结构；2)与Joan和Ron Conaway （Stowers研究所）领导的小组进行强有力的、持续的合作，他们是人类介质生物化学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.