YEAST SWI1, SWI2, AND SWI3 PROTEINS

酵母 SWI1、SWI2 和 SWI3 蛋白

• 批准号: 2187177
• 负责人: Craig L Peterson
• 金额: $ 16.51万
• 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/2187177
• 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聚合酶转录调控的机制 二.总体策略是重点关注三种酿酒酵母 基因 SWI1、SWI2 和 SWI3，编码转录因子，其 大量基因的功能需要活动 特定的激活剂。遗传学研究表明，这些 SWI 的作用之一是 产品可能是通过拮抗抑制来辅助激活剂 由核小体和其他染色质成分引起的影响。证据是 发现这些 SWI 基因在进化过程中得到了保存 多细胞真核生物，并且这些 SWI 产品是 一些发育调节因子的活动。拟议的研究可能 从而提供概念性和具体性的工具来确定有多少 真核激活剂的功能。 在博士后学习期间，我在以下方面取得了重大进展： 确定 SWI 产品在其中所扮演的角色的主要目标 转录。具体来说，已经确定SWI产品发挥 通过协助许多激活剂的功能在转录中发挥全局作用 蛋白质； SWI 产品需要协同激活 由 GAL4 转录；并且 SWI3 与糖皮质激素相关 体外受体。该提议利用了强大的遗传和 酵母中可用于检验多种假设的生化机会 关于 SWI 产品在转录中的作用。第一个目标是 该提案旨在确定协同过程中的哪些步骤 GAL4 转录的激活需要 SWI 功能。这个目标是 通过几种体内方法解决，包括体内足迹， 使用大肠杆菌 dam 甲基化酶作为染色质结构的探针，以及 使用 SWI 基因的条件等位基因的研究。第二个目标是 检验 SWI1、SWI2 和 SWI3 作为组件的假设 使用免疫共沉淀法、凝胶法形成多亚基复合物 过滤色谱法和遗传学。第三个目标是测试 假设 SWI 产品通过使用与 GAL4 激活剂相关联 生化方法和遗传筛选。第四个目标是测试 SWI1,2,3 作为新型解旋酶机的假设 确定 SWI2 解旋酶序列基序的功能作用 体内和体外。拟议研究的结果将丰富 我们了解 SWI 产品在转录中发挥的全球作用 并为监管机制提供有价值的见解 由 RNA 聚合酶 II 进行转录。