STRUCTURAL STUDIES OF EUKARYOTIC TRANSCRIPTION

真核转录的结构研究

• 批准号: 8690447
• 负责人: Francisco J Asturias
• 金额: $ 55.91万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 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 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8690447
• 关键词: Architecture; Binding; Biochemical; Biochemistry; Cell Differentiation process; Code; Collaborations; Complement; Complex; Cyclins; DNA Sequence Rearrangement; Data Collection; Development; Disease; Electron Microscopy; Enzymes; Eukaryota; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Grant; Holoenzymes; Homeostasis; Human; Image Analysis; Institutes; Malignant Neoplasms; Maps; Mediator of activation protein; Molecular; Molecular Conformation; Molecular Structure; Multiprotein Complexes; Phosphotransferases; Process; Proteins; Public Health; RNA Polymerase II; Regulation; Resolution; Role; Signal Transduction; Staging; Structure; Techniques; Transcription Initiation; Transcriptional Regulation; Work; Yeasts; base; design; expectation; experience; macromolecular assembly; macromolecule; mutant; novel; polypeptide; protein complex; public health relevance; tool; tumorigenesis

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.

描述（由申请人提供）：介体，一个大的（25个多肽，MW〜1 MDA）多蛋白质复合物在整个真核生物中保守的是负责在转录启动期间将调节信号传递到RNA聚合酶II（RNAPII）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，对介体的结构理解及其与RNAPII的相互作用对于阐明介体在启动过程中的作用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。该赠款支持的介体研究已经为复杂的分子理解奠定了基础，我们在这里描述了基于我们先前的工作的酵母和人类介体的进一步的EM，生化和功能分析，并将详细了解对介体构象和互动和互动的结构，亚基组织的结构，亚基组织，并揭示了Mediator的机械性。 在AIM 1中，我们将在亚纳光分辨率下对酵母介质（YMED）进行EM分析，并研究对YMED组装，构象变异性和功能至关重要的亚基接口。在AIM 2中，我们将研究不同因素如何控制介体构象和相互作用以调节RNAPII参与和转录起始，并获得有关介体RNAPII Holoenzyme的详细结构信息。在AIM 3中，我们将利用从YMED分析中获得的经验，从而追求对人类调解人（HMED）的结构，亚基组织和相互作用的研究。对酵母和人类介体的平行分析将揭示与跨真核生物的结构保护有关的调节机制的基本方面，同时突出了针对更复杂的人类复合物的调节方面。 我们的结果将提供对中介体能够调节转录启动的方式的分子理解，并帮助我们了解基因表达和肿瘤发生失调之间的直接联系。我们提出的工作继续我们将结构分析与生化和功能研究结合起来的策略，并将严重取决于1）与与Pawel Penczek（UT Houston）合作开发的新型EM图像分析方法的应用，专门针对研究动态复合物的结构而定制； 2）由琼和罗恩·康纳威（Joan and Ron Conaway）（斯托夫斯研究所）领导的集团进行了强有力的合作，该组织是人类调解人生化和功能研究的领导者。