Structural studies of eukaryotic transcription

描述（由申请人提供）：介体，一种在整个真核生物中保守的大的（25个多肽，MW ~1 MDa）多蛋白复合物，是负责在转录起始期间向RNA聚合酶II（RNAPII）传递调节信号的关键共激活因子。介体基本上没有酶活性，并且通常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，介体及其与RNAPII的相互作用的结构理解是必不可少的，以阐明介体的作用在启动过程中最终影响细胞分化，发育和稳态。大分子电子显微镜（EM）是表征大型动态大分子组装体的首选技术，因为它特别适合提供有关其结构，构象变化和相互作用的信息。EM研究介体的支持下，这个补助金已经设置了阶段，以实现复杂的分子理解，在这里，我们描述了进一步的EM，生化和功能分析酵母和人类介体的基础上，我们以前的工作，并将提供一个详细的了解结构，亚基组织和调节介体构象和相互作用，并揭示跨真核生物调节介体的机制。 在目的1中，我们将追求EM分析酵母介体（yMED）在亚纳米分辨率和调查亚基接口的关键yMED组装，构象变异性和功能。在目标2中，我们将研究不同的因素如何控制中介体的构象和相互作用，以调节RNAPII的参与和转录起始，并获得有关中介体-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，对人类中介（hMED）的结构、亚基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中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。 我们的研究结果将提供一个介体的方式，使转录起始的调节分子的理解，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。我们提出的工作继续我们的战略相结合的结构分析与生物化学和功能研究，并将严重依赖于1）应用新的EM图像分析方法与Pawel Penczek小组合作开发（UT休斯顿）专门为研究动态复合体的结构而量身定制; 2）与琼和罗恩科纳韦（Stowers研究所）领导的小组进行强有力的持续合作，他们是人类调解人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.