MOLECULAR MECHANISMS OF GLOBAL REPRESSION IN YEAST

描述（改编自申请人摘要）：越来越清楚 真核生物体拥有各种各样的全球转录， 抑制机制，可以控制表达的几个 其他不相关的基因簇。这些系统中的许多仍然是 对它们的了解很少，包括两个在芽殖酵母中的S.酿酒酵母， 这就是这个提案的主题。第一，不复杂， 差异地影响TATA元件的利用，因此 怀疑抑制转录的基本机制， 影响基因第二个系统利用Cyc 8-Tup 1复合物 作为转录共阻遏物被募集到受 葡萄糖氧气细胞类型和DNA损伤该提案采用 建立了分子生物学方法来阐明分子 在这两种不同的机制中， 系统，通过追求三个具体的实验路线。 在目标1中，研究了Not蛋白与以下组分的潜在相互作用： 基本的转录机制将在基因上进行测试， 生物化学提出了净化非复杂性，以检查其 组成和体外转录抑制特性。新 与Not蛋白相关或相互作用的其它蛋白质可 从双杂交和抑制子分析中出现。目标2集中于 Tup 1阻遏结构域，用一套类似于 描述了第一个（即测试与转录因子的相互作用）， 机器，体外分析和鉴定新的蛋白质， 与阻遏结构域相互作用）。目标3重点关注Cyc 8- Tup 1复合体是差异性募集的。如上所述， 描述以识别与之功能性相互作用的新蛋白质 Cyc 8和Tup 1的区域负责它们的招募， 发起人。此外，DNA结合蛋白Mig 1的区域 Cyc 8-Tup 1所需的葡萄糖抑制和Rox 1（氧抑制） 抑制将被确定，并测试与 Cyc8. 在一定程度上，参与这两个过程的分子机制 不同的全球镇压系统被揭示，知识和 其影响可能会扩展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.