YEAST SWI1, SWI2, AND SWI3 PROTEINS

描述（改编自研究者摘要）：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竞争 结构 彼得森博士的总体策略是， 保守的蛋白质复合物，SWI/SNF，这是表达一种 酵母基因的一个子集和几个转录因子的活性 活化剂。 酵母中的遗传研究表明，SWI/SNF的功能是 拮抗染色质介导的转录抑制。 过去 在预算期间，他在了解SWI/SNF如何运作方面取得了进展。 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水解的能量破坏核小体结构 在体外;评价SWI/SNF的催化重塑活性， 重组核小体阵列;并确定SWI/SNF促进了 转录激活因子与体内核小体位点的结合。 在下一个预算期间，研究人员计划继续利用 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。 第一个目标 一项提案将研究SWI/SNF在染色质转换中的作用 酵母HO基因的结构。 这一目标将由高 核小体定位和染色质的分辨率映射 使用乙酰化组蛋白的抗体的免疫沉淀测定。 的 第二个目标是检验SWI/SNF的目标是 通过基因特异性激活剂进行染色体定位。 这些研究将使用 染色质免疫沉淀法，以及体外核小体 阵列分析 第三个目标将使用定量核小体阵列测定 研究SWI/SNF作用、组蛋白 N-末端和ADA/GCN 5组蛋白乙酰转移酶。 这一目标也将考验 需要破坏组蛋白-组蛋白相互作用的假说 用于SWI/SNF催化。 第四个目标将使用SWI 2/SNF 2电池 ATP酶基序突变体和光亲和交联方法来确定 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的移动。 第五个目标将在体内使用 标记和体外缔合试验，以研究亚基 SWI/SNF组织。

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.