Analysis of General Transcription Factors in Yeast

描述(申请人提供)：本申请的长期目标是更多地了解真核生物转录起始的基本和保守方面。这些研究将集中于在酵母中发现的一种蛋白质复合体，它控制着RNA聚合酶II的转录启动。这个复合体被称为SAGA(SPT-Ada-Gcns5乙酰转移酶)，是一类被称为辅助激活因子的因子的成员，该因子在转录启动控制中发挥关键作用。共激活子是一种大的多蛋白复合体，通常具有多种活性，在某些情况下既能激活又能抑制转录。佐贺是保守的，因为人类佐贺样复合体已经被发现。关于辅助激活子的几个问题，包括它们的激活机制，不同辅助激活子之间的功能冗余，以及一些辅助激活子如何同时激活和抑制转录，目前还知之甚少。 拟议的实验是为了研究SAGA在酵母中的几个功能问题。具体目标1包括三套实验。前两组利用生化和遗传学方法研究了SAGA组分Spt3的激活机制。第三组涉及一些SAGA亚基的乙酰化可能的作用。具体目标2涉及SAGA活性与其他共激活剂复合体的体内协调。第一部分将遗传分析与微阵列研究相结合，以解决SAGA和第二个辅激活子复合体Swi/Snf之间的功能冗余。第二部分旨在确定与SAGA一起作用的其他因素，以激活研究得很好的GAL1启动子的转录。特定目标3将重点转移到转录抑制问题上。MOT3是一种与SAGA功能相关的DNA结合抑制因子。Mot3在抑制合成麦角固醇所需的基因中起着关键作用，麦角固醇是酿酒酵母细胞膜的关键成分。这些基因的强烈抑制也需要较低的氧气水平。提出了两套实验方案。首先，将讨论抑制的机制，检验SAGA也是抑制这些基因所必需的初步发现，确定其他必要的因素，并阐明它们各自的作用。其次，将使用分子和遗传分析来解决氧介导的调节的作用。 这些研究应该揭示酵母转录调控的重要方面。鉴于转录机制的高度保守性，这一结果将适用于理解人类的转录。

DESCRIPTION (provided by applicant): 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 cervisiae, that controls transcription initiation by RNA polymerase II. This complex, named SAGA (Spt-Ada-Gcns5 Acetyltransferase), is a member of a class of factors, called coactivators that play critical roles in the control of transcription initiation. Coactivators are large, multiprotein 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 experiments. 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 GAL1 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 addresses 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.