YEAST SWI1, SWI2, AND SWI3 PROTEINS

描述(改编自调查人员的摘要)：总体 这项研究的目的是确定染色体结构如何影响 基因表达和转录机制如何与之抗衡 结构。彼得森博士的总体策略是专注于进化上的 保守的蛋白质复合体，SWI/SNF，这是表达 酵母基因亚群和几种转录因子的活性 激活剂。酵母中的遗传学研究表明SWI/SNF通过以下途径发挥作用 拮抗染色质介导的转录抑制。在过去的时间里 在预算期间，他在理解安全倡议/国家安全部队如何运作方面取得了进展。 特别是，他从酵母中纯化了SWI/SNF复合体；证明了 SWI/SNF可以利用ATP水解的能量来破坏核小体结构 体外实验：评价SWI/SNF对血管内皮的催化重塑活性 重组核小体阵列；并确定SWI/SNF有助于 体内转录激活剂与核小体结合。 在下一个预算期间，调查员计划继续利用 酵母中强大的遗传和生化机会可用于 研究SWI/SNF在体内的作用及其生化机制 其中SWI/SNF在体外破坏核小体结构。的第一个目标是 提案将研究SWI/SNF在染色质转变中的作用 酵母HO基因在体内的结构。这一目标将由HIGH 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试验。这个 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 阵列法。第三个目标将使用定量核小体阵列分析 探讨SWI/SNF作用与组蛋白的关系 N末端和ADA/GCN5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设 用于SWI/SNF催化。第四个目标将使用SWI2/SNF2电池 ATPase基序突变体和光亲和交联法确定 ATP结合和水解在SWI/SNF重塑中的作用。这一目标将 也使用荧光方法来检验SWI/SNF作用的假设 导致核小体DNA的运动。第五个目标将在体内使用 标记和体外结合实验研究亚基 SWI/SNF的组织。

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.