YEAST SWI1, SWI2, AND SWI3 PROTEINS

酵母 SWI1、SWI2 和 SWI3 蛋白

• 批准号: 3308809
• 负责人: Craig L Peterson
• 金额: $ 17.76万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3308809
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA footprinting; Saccharomyces cerevisiae; alleles; chromatin; fungal genetics; gel filtration chromatography; genetic transcription; immunoprecipitation; nucleic acid sequence; plant proteins; protein structure; transcription factor

The overall objective of this work is to determine the molecular mechanisms that govern the regulation of transcription by RNA polymerase II. The general strategy is to focus on three Saccharomyces cerevisiae genes, SWI1, SWI2, and SWI3, which encode transcription factors whose activities are required for the function of a large number of gene- specific activators. Genetic studies indicate that one role for these SWI products may be to assist activators by antagonizing the inhibitory effects caused by nucleosomes and other chromatin components. Evidence is emerging that these SWI genes have been conserved during the evolution of multi-cellular eukaryotes and that these SWI products are required for the activity of several developmental regulators. The proposed studies may thus provide tools, both conceptual and concrete, for determining how many eukaryotic activators function. During my postdoctoral studies, significant progress has been made in my primary goal of determining the role that SWI products play in transcription. Specifically, it has been determined that SWI products play a global role in transcription by assisting the function of many activator proteins; that SWI products are required for synergistic activation of transcription by GAL4; and that SWI3 is associated with the glucocorticoid receptor in vitro. This proposal exploits the powerful genetic and biochemical opportunities available in yeast to test several hypotheses concerning the role of SWI products in transcription. The first aim of this proposal is to determine which steps in the process of synergistic activation of transcription by GAL4 require SWI function. This aim is addressed by several in vivo methods, that include in vivo footprinting, the use of E. coli dam methylase as a probe of chromatin structure, and studies that use conditional alleles of SWI genes. The second objective is to test the hypothesis that SWI1, SWI2, and SWI3 function as components of a multisubunit complex by using coimmunoprecipitation assays, gel filtration chromatography, and genetics. The third aim is to test the hypothesis that SWI products associate with the GAL4 activator by using biochemical methods and a genetic screen. The fourth objective is to test the hypothesis that SWI1,2,3 functions as a novel helicase machine by determining the functional role of the SWI2 helicase sequence motifs both in vivo and in vitro. The results from the proposed studies will enrich our knowledge of the global role that SWI products play in transcription and provide valuable insights into the mechanisms for regulating transcription by RNA polymerase II.

这项工作的总体目标是确定分子 RNA聚合酶调控转录的机制 二.总的策略是集中在三个酿酒酵母 基因SWI 1、SWI 2和SWI 3，其编码转录因子， 活性是大量基因的功能所必需的， 特定的激活剂。遗传学研究表明，这些SWI的一个作用是， 产品可能是通过拮抗抑制剂， 由核小体和其他染色质成分引起的作用。证据 这些SWI基因在进化过程中是保守的， 多细胞真核生物，这些SWI产品是必需的， 几种发育调节剂的活性。拟议的研究可 从而提供了概念上和具体的工具， 真核生物激活剂的功能。 在我的博士后研究期间，我的研究取得了重大进展。 主要目标是确定SWI产品在 转录。具体而言，已确定SWI产品发挥 通过协助许多激活因子的功能在转录中发挥全局作用 蛋白质; SWI产品是协同激活 GAL 4转录; SWI 3与糖皮质激素相关 体外受体。这项提案利用了强大的遗传和 酵母中可用的生化机会来测试几种假设 关于SWI产物在转录中的作用。的首要目标 该建议旨在确定协同过程中的哪些步骤， GAL 4激活转录需要SWI功能。该目的是 通过几种体内方法，包括体内足迹法， 利用E. coli dam甲基化酶作为染色质结构的探针，和 使用SWI基因的条件等位基因的研究。第二个目标是 为了检验SWI 1、SWI 2和SWI 3作为 用免疫共沉淀法检测多亚基复合物， 过滤层析和遗传学。第三个目标是测试 假设SWI产物与GAL 4激活剂相关， 生物化学方法和遗传筛选。第四个目标是测试 SWI 1，2，3作为一种新型解旋酶机器的假设， 确定SWI 2解旋酶序列基序的功能作用， 在体内和体外。拟议研究的结果将丰富 我们对SWI产品在转录中发挥的全球作用的了解 并提供有价值的见解， 通过RNA聚合酶II转录。