The Role of Yeast Mediator in Transcriptional Regulation

RNA 聚合酶 II (pol II) 转录的调节控制所有细胞过程。 因此，该过程的外部控制是治疗剂的一个有吸引力的目标。 该提案重点关注与启动子结合的多种调控因子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，染色质抑制的解除，随后聚合酶和辅助因子与启动子相互作用以开始转录。转录抑制子和激活子调节每个步骤的启动。 拟议的研究将探讨酿酒酵母介体（所有真核细胞中发现的一种全局共激活剂）在转录起始组装步骤调节中的作用。 我们研究的长期目标是使用多学科方法在也包含染色质的重建系统中研究分子水平上的转录激活和抑制。 由于转录及其调控的基本机制从酵母到人类基本上都是保守的，因此我们的研究将提供对基因表达的一般见解。 新兴的哺乳动物遗传学研究将不同形式的介体与疾病状态联系起来，强调了了解介体复合体的基本机制的迫切需要。我们的研究基于高度纯化的酵母转录系统，其中激活剂需要介体来刺激反应。 体外系统的结果需要经过酵母遗传学的验证。我们当前的目标是仔细剖析酵母Mediator的机制。 本研究的具体目标是：首先，通过分析酵母同源物来确定所有后生动物介体高度保守的核心亚基的功能。 我们将从酵母中去除最广泛使用的亚基 Med7p 开始，并使用基因组、生化和遗传学方法来评估该蛋白质的作用。 其次，纯化调节介体活性的因子，并在高度纯化的酵母转录系统中重建这些因子与介体的生理相互作用。 在一种情况下，将表征特异性抑制介体促进的激活转录的因子和该抑制剂的拮抗剂。 在另一种情况下，我们将通过生化表征介体拮抗的转录一般阻遏物来确定为什么体内所有转录都需要介体。

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.