YEAST SWI1, SWI2, AND SWI3 PROTEINS

酵母 SWI1、SWI2 和 SWI3 蛋白

• 批准号: 2703771
• 负责人: Craig L Peterson
• 金额: $ 26.43万
• 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 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2703771
• 关键词: DNA binding protein; Saccharomyces cerevisiae; acetylation; acyltransferase; adenosine triphosphate; chromatin; chromosomes; crosslink; fluorescence spectrometry; fungal genetics; genetic transcription; histones; hydrolysis; immunoprecipitation; nucleic acid sequence; nucleosomes; protein structure

DESCRIPTION (adapted from the investigator's abstract): The overall objective of this research is to determine how chromosome structure affects gene expression and how the transcription machinery contends with this structure. Dr. Peterson's general strategy is to focus on an evolutionarily conserved protein complex, SWI/SNF, which is required for expression of a subset of yeast genes and for the activity of several transcriptional activators. Genetic studies in yeast suggest that SWI/SNF functions by antagonizing chromatin-mediated transcriptional repression. Over the past budget period he has made progress in understanding how SWI/SNF functions. In particular, he purified SWI/SNF complex from yeast; demonstrated that SWI/SNF can use the energy of ATP hydrolysis to disrupt nucleosome structure in vitro; evaluated the catalytic remodeling activity of SWI/SNF on reconstituted nucleosome arrays; and determined that SWI/SNF facilitates the binding of a transcriptional activator to a nucleosomal site in vivo. Over the next budget period the investigator plans to continue to exploit the powerful genetic and biochemical opportunities available in yeast to investigate the role of SWI/SNF in vivo and the biochemical mechanism by which SWI/SNF disrupts nucleosome structure in vitro. The first aim of the proposal will investigate the role of SWI/SNF in transitions in chromatin structure of the yeast HO gene in vivo. This aim will be addressed by high resolution mapping of nucleosome positioning and a chromatin immunoprecipitation assay that uses antibodies to acetylated histones. The second objective is to test the hypothesis that SWI/SNF is targeted to chromosomal locations by gene-specific activators. These studies will use a chromatin immunoprecipitation method, as well as an in vitro nucleosome array assay. The third aim will use a quantitative nucleosome array assay to investigate the relationship between SWI/SNF action, the histone N-termini, and ADA/GCN5 histone acetyltransferases. This aim will also test the hypothesis that disruption of histone-histone interactions is required for SWI/SNF catalysis. The fourth objective will use a battery of SWI2/SNF2 ATPase motif mutants and a photoaffinity crosslinking method to define the role of ATP binding and hydrolysis in SWI/SNF remodeling. This aim will also use fluorescence methods to test the hypothesis that SWI/SNF action causes movements of nucleosomal DNA. The fifth aim will use in vivo labeling and in vitro association assays to investigate the subunit organization of SWI/SNF.

描述（改编自研究者的摘要）：总体 这项研究的目的是确定染色体结构如何影响 基因表达以及转录机制如何与之抗衡 结构。 彼得森博士的总体策略是关注进化论 保守的蛋白质复合物，SWI/SNF，它是表达 酵母基因的子集和几个转录活性 激活剂。 酵母基因研究表明 SWI/SNF 的功能是通过 拮抗染色质介导的转录抑制。 过去的事 预算期间，他在了解 SWI/SNF 的运作方式方面取得了进展。 特别是，他从酵母中纯化了 SWI/SNF 复合物；证明了 SWI/SNF可以利用ATP水解的能量来破坏核小体结构 体外;评估了 SWI/SNF 的催化重塑活性 重建核小体阵列；并确定 SWI/SNF 有助于 转录激活剂与体内核小体位点的结合。 在下一个预算期间，调查人员计划继续利用 酵母中强大的遗传和生化机会 研究SWI/SNF在体内的作用及其生化机制 SWI/SNF 在体外破坏核小体结构。 该项目的第一个目标是 该提案将研究 SWI/SNF 在染色质转变中的作用 酵母HO基因的体内结构。 这一目标将由高层来实现 核小体定位和染色质的分辨率作图 使用乙酰化组蛋白抗体的免疫沉淀测定。 这 第二个目标是检验 SWI/SNF 的目标假设 基因特异性激活剂的染色体位置。 这些研究将使用 染色质免疫沉淀法以及体外核小体 阵列测定。 第三个目标将使用定量核小体阵列测定 为了研究 SWI/SNF 作用、组蛋白之间的关系 N 末端和 ADA/GCN5 组蛋白乙酰转移酶。 这个目标也将考验 需要破坏组蛋白-组蛋白相互作用的假设 用于 SWI/SNF 催化。 第四个目标将使用 SWI2/SNF2 电池 ATP酶基序突变体和光亲和交联方法来定义 ATP 结合和水解在 SWI/SNF 重塑中的作用。 这一目标将 还使用荧光方法来检验 SWI/SNF 作用的假设 引起核小体 DNA 的运动。 第五个目标将在vivo使用 通过标记和体外关联分析来研究亚基 SWI/SNF 的组织。