MOLECULAR MECHANISMS OF GLOBAL REPRESSION IN YEAST

描述(改编自申请人的摘要)：情况正在变得明朗 真核生物拥有各种各样的全球转录 可以控制几个基因表达的抑制机制 原本互不相关的基因簇。这些系统中的许多仍然 人们对此知之甚少，包括在萌芽酵母中的两个，酿酒酵母， 这些都是这项提案的主题。一是不复杂的， 不同地影响塔塔元素的利用率，因此 怀疑抑制了转录的基本机制 受影响的基因。第二个系统利用Cyc8-Tup1复合体 作为转录共抑制子招募到受 葡萄糖、氧气、细胞类型和DNA损伤。该提案雇用了 已建立的分子生物学方法来阐明分子 在这两部影片中，压制的细节有很大的不同 系统，通过追求三个具体的实验路线。 在目标1中，NOT蛋白与组分的潜在相互作用 基本的转录机制将进行基因测试和 生化方面的。提出提纯非复合体，考察其 组成和体外转录抑制特性。新的 与NOT蛋白相关或与NOT蛋白相互作用的其他蛋白质可以 来自双杂交和抑制者的分析。Aim 2专注于 Tup1抑制域，有一组类似的方法 描述了第一个(即与转录的相互作用的测试 机械，体外分析和鉴定新的蛋白质， 与抑制域相互作用)。目标3重点介绍Cyc8如何- Tup1复合体是差异性招募的。如上所述，方法有 被描述为识别与之功能相互作用的新蛋白质 Cyc8和Tup1地区负责招募他们到特定的 推动者。此外，DNA结合蛋白Mig1的区域 (葡萄糖抑制)和Rox1(氧气抑制)是Cyc8-Tup1所必需的 将确定压制，并测试其直接交互作用 Cyc8。 在某种程度上，这两个过程中涉及的分子机制 不同的全球压制体系被揭示出来，知识和 其影响可能延伸到整个真核生物领域。 有机体。

DESCRIPTION (Adapted from applicant's abstract): It is becoming clear that eukaryotic organisms possess a variety of global transcriptional repression mechanisms that can control the expression of several clusters of otherwise unrelated genes. Many of these systems are still poorly understood, including two in the budding yeast, S. cerevisiae, that are the topic of this proposal. One, the Not complex, differentially affects TATA element utilization, and is therefore suspected to inhibit the basic machinery of transcription of the affected genes. The second system utilizes the Cyc8-Tup1 complex recruited as a transcriptional co-repressor to promoters regulated by glucose, oxygen, cell-type, and DNA damage. The proposal employs established molecular biological approaches to elucidate the molecular details of repression in these two rather mechanistically different systems, by pursuing three specific lines of experimentation. In Aim 1, potential interactions of the Not proteins with components of the basic transcriptional machinery will be tested genetically and biochemically. Purifying the Not complex is proposed, to examine its composition and transcriptional repression character in vitro. New additional proteins related to or interacting with the Not proteins may emerge from two-hybrid and suppressor analysis. Aim 2 concentrates on the Tup1 repression domain, with a set of approaches similar to that described for the first (i.e. tests for interaction with transcriptional machinery, in vitro analysis, and identification of new proteins that interact with the repression domain). Aim 3 focuses on how the Cyc8- Tup1 complex is differentially recruited. As above, approaches are described to identify new proteins that functionally interact with regions of Cyc8 and Tup1 responsible for their recruitment to specific promoters. Moreover, the regions of the DNA-binding proteins Mig1 (glucose repression) and Rox1 (oxygen repression) required for Cyc8-Tup1 repression will be determined, and tested for direct interaction with Cyc8. To the extent that the molecular mechanisms involved in these two distinct global repression systems are revealed, the knowledge and implications are likely to extend throughout the domain of eukaryotic organisms.