STRUCTURE/REGULATION OF THE YEAST HSP90 GENES

酵母 HSP90 基因的结构/调控

• 批准号: 2022461
• 负责人: David Samuel Gross
• 金额: $ 14.5万
• 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/2022461
• 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签申请旨在解决 转录激活剂如何刺激体内启动子活性。 在 酿酒酵母，热休克因子（HSF）激活 至少两个热休克基因——HSP82和HSC82——的转录 减轻对其上游调控区域的核小体抑制。 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，这是必不可少的初始步骤 转录激活。 格罗斯博士建议识别激活 酵母 HSF (yHSF) 的结构域指导体内染色质重塑。 首先，使用基因抑制测定，研究人员希望选择 核小体。 对这种显性 HSF1 抑制因子进行测序应该允许 识别原生重塑域；互补的 HSF1 结构域 将进行删除分析以确认这一分配。 第二，博士。 格罗斯提出表征结构和功能表型 与 HSF1 突变体相关，并确定它们在两者中的作用 在目标温度下建立和维持无核小体表型 冲击促进剂。 三是着力解决改造机制问题 域有效，申请人将生化重组 HSP82 使用纯化的组分将启动子转化为稳定的双核小体复合物。 将尝试使用 HeLa 和酿酒酵母核心进行重构 组蛋白，目标是形成完整的同源系统。 格罗斯博士随后将 询问重组 yHSF 是否可以结合并破坏重组的 复杂，无论这种活动是通过存在来介导（或增强）的 SWI/SNF 复合物和 ATP，是否因 hsp70 的存在而减弱 或突变组蛋白，以及功能获得性 yHSF 突变多肽是否是 体外重塑比野生型 yHSF 更有效。 最后， 申请人将推定的 yHSF 重塑域的能力 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自以下事实 许多人类疾病，包括许多癌症，都与 启动子特异性转录因子中存在错义突变。 此外，已知 SWI/SNF 复合物与以下蛋白质相关： 增强或抑制人类肿瘤的形成。