Analysis of General Transcription Factors in Yeast

这个应用程序的长期目标是学习更多关于基础和守恒的知识 真核生物转录启动的几个方面。这些研究将集中在发现的一种蛋白质复合体上 酿酒酵母一种酵母，酿酒酵母，控制RNA聚合酶I1的转录启动这 复合体，命名为SAGA(SPT-Ada-Gcn5乙酰转移酶)，是一类被称为 共激活因子，在转录启动的控制中起着关键作用。共激活剂是大型的，多个- 通常具有多种活性的蛋白质复合体，在某些情况下既能激活又能抑制 抄写。佐贺是保守的，因为人类佐贺样复合体已经被发现。几个问题 关于共激活剂，包括它们的激活机制，功能 不同辅助激活剂之间的冗余，以及一些辅助激活剂如何既激活又抑制 抄写。 拟议的实验是为了研究SAGA在酵母中的几个功能问题。特定的 目标1包含三组实验。前两个集合解决了由 SAGA组分Spt3，使用生化和遗传方法。第三套解决了可能的 某些SAGA亚基的乙酰化作用。特定目标2解决了SAGA的体内协调 与其他共激活剂络合物的活性。第一部分将遗传分析与基因芯片相结合 研究解决SAGA和第二个共激活复合体Swi/Snf之间的功能冗余。 第二部分旨在确定与SAGA一起发挥作用的其他因素，以激活转录 研究很好的GAL-1启动子。特定目标3将重点转移到转录抑制问题上。Mot3是 一种在功能上与SAGA相关的DNA结合抑制因子。Mot3在抑制 合成麦角甾醇所需的基因，麦角固醇是酿酒酵母细胞膜的关键成分。强大的镇压 这些基因中的大多数也需要低氧水平。提出了两套实验方案。首先， 压抑的机制将被解决，测试的初步发现，佐贺也需要 抑制这些基因，确定其他必要的因素，并阐明它们各自的作用。 其次，氧介导的调节作用将通过分子和遗传分析来解决。 这些研究应该揭示酵母转录调控的重要方面。考虑到强者 转录机制的保守性，结果将适用于理解转录。 人类。

The long-term objectives of this application are to learn more about fundamental and conserved aspects of transcription initiation in eukaryotes. The studies will focus on a protein complex discovered in the yeast, Saccharomyces cerevisiae, that controls transcription initiation by RNA polymerase I1. This complex, named SAGA (Spt-Ada-Gcn5 Acetyltransferase), is a member of a class of factors, called coactivators, that play critical roles in the control of transcription initiation. Coactivators are large, multi- protein complexes that often possess multiple activities and in some cases can both activate and repress transcription. SAGA is conserved, as human SAGA-like complexes have been identified. Several issues about coactivators are poorly understood, including their mechanisms of activation, the functional redundancy between different coactivators, and how some coactivators can both activate and repress transcription. The proposed experiments are to study several issues concerning SAGA's functions in yeast. Specific Aim 1 contains three sets of experments. The first two sets address the mechanism of activation by the SAGA component, Spt3, using biochemical and genetic approaches. The third set addresses the possible role of acetylation of some SAGA subunits. Specific Aim 2 addresses the in vivo coordination of SAGA's activities with other coactivator complexes. The first section combines genetic analysis with microarray studies to address the functional redundancy between SAGA and a second coactivator complex, Swi/Snf. The second section is designed to identify other factors that function with SAGA to activate transcription of the well-studied GAL 1 promoter. Specific Aim 3 shifts focus to address repression of transcription. Mot3 is a DNA-binding repressor that is functionally related to SAGA. Mot3 plays a critical role in the repression of genes required for synthesis of ergosterol, a key component of S. cerevisiae membranes. Strong repression of these genes also requires low oxygen levels. Two sets of experiments are proposed. First, the mechanism of repression will be addressed, testing the preliminary finding that SAGA is also required for repression of these genes, identifying other factors necessary, and elucidating their respective roles. Second, the role of oxygen-mediated regulation will be addressed using molecular and genetic analysis. These studies should reveal important aspects of transcriptional control in yeast. Given the strong conservation of transcriptional mechanisms, the results will be applicable to understanding transcription in humans.