STRUCTURE/REGULATION OF THE YEAST HSP90 GENES

酵母 HSP90 基因的结构/调控

• 批准号: 2900750
• 负责人: David Samuel Gross
• 金额: $ 15.72万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900750
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA footprinting; HeLa cells; Saccharomyces cerevisiae; Xenopus; chromatin; environmental stressor; fungal genetics; gene deletion mutation; gene expression; gene interaction; genetic promoter element; genetic regulatory element; genetic transcription; northern blottings; nucleic acid sequence; site directed mutagenesis; stress proteins; structural genes; transcription factor

DESCRIPTION: This renewal application by Dr. Gross is designed to address how transcriptional activators stimulate promoter activity in vivo. In the yeast Saccharomyces cerevisiae, heat shock factor (HSF) activates transcription of at least two heat shock genes--HSP82 and HSC82-- by alleviating nucleosomal repression of their upstream regulatory regions. Unknown is how this protein--or any protein-- mediates local disruption of unfolding of promoter chromatin structure, the essential initial step of transcriptional activation. Dr. Gross proposes to identify the activation domains of yeast HSF (yHSF) that direct chromatin remodeling in vivo. First, using a genetic suppression assay, the investigator wishes to select nucleosomes. Sequencing of such dominant HSF1 suppressors should permit identification of the native remodeling domain; a complementary HSF1 domain deletion analysis will be conducted to confirm this assignment. Second, Dr. Gross proposes to characterize structural and functional phenotypes associated with the HSF1 mutants, and ascertain their role in both establishing and maintaining the nucleosome-free phenotype at a target heat shock promoter. Third, to address the mechanism by which the remodeling domain works, the applicant will biochemically reconstitute the HSP82 promoter into a stable dinucleosomal complex using purified components. Reconstitution will be attempted using both HeLa and S. cerevisiae core histone, with the goal being a full homologous system. Dr. Gross will then ask whether recombinant yHSF can bind and disrupt this reconstituted complex, whether this activity is mediate (or enhanced) by the presence of SWI/SNF complex and ATP, whether it is attenuated by the presence of hsp70 or mutant histone, and whether gain-of-function yHSF mutant polypeptides are more effective in in vitro remodeling that wild-type yHSF. Finally, the applicant will the ability of the putative yHSF remodeling domain to interact with SWI/SNF subunits, as well as individual core histone and components of the general transcription complex, by performing GST-pull down experiments. The health relatedness of this project derives from the fact that a large number of human diseases, including many cancers, correlate with missense mutations in promoter-specific transcription factors. Moreover, the SWI/SNF complex is known to associate with proteins that either enhance or suppress the formation of human tumors.

描述：格罗斯博士的这份续期申请旨在解决 转录激活因子如何在体内刺激启动子活性。 在 酵母酿酒酵母，热休克因子（HSF）激活 至少两个热休克基因-HSP 82和HSC 82-的转录， 减轻其上游调控区的核小体抑制。 未知的是这种蛋白质-或任何蛋白质-如何介导局部破坏， 启动子染色质结构的展开， 转录激活 格罗斯博士建议确定 酵母HSF（yHSF）的结构域，其在体内指导染色质重塑。 首先，使用遗传抑制测定，研究者希望选择 核小体 这种显性HSF 1抑制子的测序应该允许 天然重塑结构域的鉴定;互补HSF 1结构域 将进行删除分析以确认该分配。 第二，博士。 Gross建议将结构和功能表型 与HSF 1突变体相关，并确定它们在两种疾病中的作用 在目标温度下建立和维持无核小体表型 休克促进剂。 第三，解决重塑的机制问题， 域作品，申请人将生化重组HSP 82 启动子转化为稳定的双核体复合物。 将尝试使用HeLa和S进行复溶。酿酒酵母核心 组蛋白，目标是一个完整的同源系统。 格罗斯医生会 问重组yHSF是否可以结合并破坏这种重组的 复杂，无论这种活动是介导（或增强）的存在， SWI/SNF复合物和ATP，无论其是否因hsp 70的存在而减弱 或突变组蛋白，以及功能获得性yHSF突变多肽是否 在体外重塑中比野生型yHSF更有效。 最后 申请人将假定的yHSF重塑结构域的能力， 与SWI/SNF亚基以及单个核心组蛋白相互作用， 一般转录复合物的组成部分，通过执行GST下拉 实验 该项目的健康相关性源于以下事实 许多人类疾病，包括许多癌症， 启动子特异性转录因子中有错义突变。 此外，已知SWI/SNF复合物与 增强或抑制人类肿瘤的形成。