Mechanism of transcription regulation by the Mediator

介体的转录调控机制

基本信息

批准号：
8961821
负责人：
Yuichiro Takagi
金额：
$ 30.59万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8961821
关键词：
Address Affect Architecture Back Binding Sites Biochemical Biological Biological Assay Boxing Complex DNA DNA Polymerase II Data Electron Microscope Electrons Engineering Ensure Eukaryota Exhibits Future Gene Expression General Transcription Factors Generations Genes Genetic Genetic Transcription Head Health Human Image In Vitro Investigation Knowledge Lead Light Link Malignant Neoplasms Mass Spectrum Analysis Mediator of activation protein Mental disorders Methods Molecular Conformation Molecular Machines Mutation Play Polymerase Public Health RNA Polymerase II Recombinants Recruitment Activity Resolution Role Saccharomyces cerevisiae Site Structure System Tail Technology Testing Time Titan Transcription Coactivator Transcription Initiation Transcription Process Transcriptional Activation Transcriptional Regulation Work Yeasts detector empowered human disease in vivo malignant neurologic neoplasms molecular mass movie nervous system disorder particle promoter protein complex protein expression public health relevance reconstitution stoichiometry transcription factor

项目摘要

DESCRIPTION (provided by applicant): Mediator is an essential regulator of eukaryotic transcription that orchestrates transcription factors and RNA polymerase II (Pol II) assembly onto a gene promoter to facilitate transcription initiation. Mutations in several human Mediator subunits have been linked to neurological disorders and cancers, exemplifying the importance of proper Mediator function in human health. Yet, how Mediator orchestrates transcription is still a big black box. The structural complexity of Mediator has been a roadblock for structure-function studies that attempt to shed light on that black box. We have developed the experimental system that overcomes this roadblock by capturing and visualizing "Mediator in action" during transcription, thereby determining the transcription activation mechanisms by Mediator. Mediator from yeast is composed of 21 subunits with a molecular mass over 1 MDa. Despite of significant recent progress, the structure of the most biological relevant form of Mediator, when it is in the context of active transcription initiation (e.g. DNA-activator-Mediator has not been available because of two primary limitations: 1) lack of well-defined recombinant forms of the Mediator complex; and 2) lack of biochemical system to reconstitute the Mediator in active transcription form in vitro. To overcome these two limitations, we first developed a new protein expression technology that enables us to express the entire 21 subunits of Mediator. Second, we implemented the in vitro system in which the transcription process with Mediator can be recapitulated. Combining these two new methods with cryo-EM and single-particle analysis allows us to determine the mechanism of how Mediator orchestrates the assembly of transcription factors and Pol II onto promoter DNA for transcription initiation (Aim 1), to determine the structure of the 21-subunit Mediator in the context of transcription by cryo-EM and single-particle analysis (Aim 2), and to examine genetic interaction between Mediator and Pol II in vivo (Aim 3). Successful completion of the proposed project will reveal the fundamental mechanisms of Mediator in transcription regulation. The knowledge gained from yeast Mediator will be applied to that of higher eukaryotes including humans. Furthermore, our experimental strategy will be applied to that of human system in the future.

描述（由申请人提供）：介体是真核转录的重要调节因子，它协调转录因子和 RNA 聚合酶 II (Pol II) 组装到基因启动子上，以促进转录起始。几个人类介体亚基的突变与神经系统疾病和神经系统疾病有关。然而，Mediator 的结构复杂性一直是结构功能研究的障碍。我们开发了一个实验系统，通过捕获和可视化转录过程中的“中介体”来克服这个障碍，从而确定来自酵母的中介体由 21 个亚基组成的转录激活机制。分子量超过 1 MDa。尽管最近取得了重大进展，但在主动转录起始的情况下，生物学上最相关的介体形式的结构（例如 DNA 激活剂-介体）尚未获得，因为有两个主要限制： 1）缺乏明确的介体复合物重组形式；2）缺乏在体外以活性转录形式重建介体的生化系统为了克服这两个限制，我们首先开发了一种新的蛋白质表达技术，使我们能够其次，我们实现了体外系统，将这两种新方法与冷冻电镜和单粒子分析相结合，可以重现介体的转录过程。 Mediator 如何协调转录因子和 Pol II 组装到启动子 DNA 上以进行转录起始的机制（目标 1），通过冷冻电镜和单粒子分析确定转录背景下 21 个亚基 Mediator 的结构（目标2），并检查介体和 Pol II 之间的体内遗传相互作用（目标 3）。成功完成拟议项目将揭示介体在转录调控中的基本机制，从酵母介体中获得的知识将应用于更高级的介体。此外，我们的实验策略将来还将应用于人类系统。