The Role of Yeast Mediator in Transcriptional Regulation

酵母介体在转录调控中的作用

• 批准号: 6768577
• 负责人: Lawrence Christopher Myers
• 金额: $ 24.58万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6768577
• 关键词: DNA binding protein; DNA directed RNA polymerase; Saccharomyces cerevisiae; cell free system; chromatin; chromatography; enzyme complex; enzyme inhibitors; enzyme reconstitution; fungal genetics; fungal proteins; gene induction /repression; genetic regulation; genetic regulatory element; genetic transcription; immunologic assay /test; immunoprecipitation; intermolecular interaction; mass spectrometry; microarray technology; molecular assembly /self assembly; protein purification; protein structure function; stoichiometry; suppressor mutations; temperature sensitive mutant

Regulation of RNA polymerase II (pol II) transcription controls all cellular processes. As a result, the external control of this process is an attractive target for therapeutic agents. This proposal focuses on the mechanisms by which the activation potential of diverse regulatory factors bound to a promoter translates into increased activity of the core pol II transcription machinery on its target gene. Transcription initiation proceeds in two stages, the relief of inhibition by chromatin followed by the interaction of polymerase and accessory factors with the promoter to start transcription. Transcriptional repressors and activators regulate initiation at each of these steps. The proposed research will address the role of Saccharomyces cerevisiae Mediator, a global co-activator found in all eukaryotic cells, in regulation of the assembly step of transcription initiation. The long range goal of our research is to use a multi-disciplinary approach to investigate transcriptional activation and repression on a molecular level in a reconstituted system that also incorporates chromatin. Since the underlying mechanisms of transcription and its regulation are fundamentally conserved from yeast to man, our studies will provide general insight into gene expression. Emerging mammalian genetic studies, which associate different forms of Mediator with disease states, accentuate the imperative need to understand the fundamental mechanisms of Mediator complexes. Our studies are based on a highly purified yeast transcription system in which Mediator is required for activators to stimulate the reaction. The results of the in vitro system are subject to validation by yeast genetics. Our current goal is to carefully dissect the mechanism of yeast Mediator. The specific objectives of this research are: First, to ascertain the function of the highly conserved core subunits of all metazoan Mediators by analyzing the yeast homologs. We will start by depleting Med7p, the most widely utilized subunit, from yeast and using genomic, biochemical and genetic methods to assess the role of this protein. Second, to purify factors that modulate Mediator activity and reconstitute the physiological interplay of these factors with Mediator in the highly purified yeast transcription system. In one case a factor that specifically inhibits Mediator facilitated activated transcription and the antagonist of this inhibitor will be characterized. In the other case we will determine why Mediator is required for all transcription in vivo by biochemically characterizing a general repressor of transcription antagonized by Mediator.

RNA聚合酶II （pol II）的转录调控控制着所有的细胞过程。因此，这一过程的外部控制是治疗剂的一个有吸引力的目标。本研究的重点是不同调控因子与启动子结合的激活潜力转化为核心pol II转录机制在其靶基因上的活性增加的机制。转录起始分两个阶段进行，染色质抑制解除后，聚合酶和辅助因子与启动子相互作用开始转录。转录抑制因子和激活因子调节这些步骤的起始。本研究将探讨酿酒酵母（Saccharomyces cerevisiae Mediator）在转录起始组装过程中的调控作用。酿酒酵母是一种存在于所有真核细胞中的全局共激活因子。我们研究的长期目标是使用多学科方法在分子水平上研究含有染色质的重组系统中的转录激活和抑制。由于从酵母到人类的转录及其调控的潜在机制基本上是保守的，我们的研究将为基因表达提供一般的见解。新兴的哺乳动物遗传学研究将不同形式的介质与疾病状态联系起来，强调了理解介质复合物基本机制的迫切需要。我们的研究是基于一个高度纯化的酵母转录系统，其中需要介质的激活剂来刺激反应。体外系统的结果有待酵母遗传学的验证。我们目前的目标是仔细剖析酵母介质的机制。本研究的具体目标是：首先，通过分析酵母同源物，确定所有后生动物介质中高度保守的核心亚基的功能。我们将首先从酵母中提取最广泛使用的亚基Med7p，并使用基因组学、生化和遗传学方法来评估该蛋白的作用。其次，纯化调节Mediator活性的因子，并在高度纯化的酵母转录系统中重建这些因子与Mediator的生理相互作用。在一种情况下，特异性抑制中介促进激活转录的因子和这种抑制剂的拮抗剂将被表征。在另一种情况下，我们将确定为什么所有的转录都需要中介体，通过生物化学表征被中介体拮抗的一般转录抑制因子。