STRUCTURE/REGULATION OF THE YEAST HSP90 GENES

酵母 HSP90 基因的结构/调控

• 批准号: 2684964
• 负责人: David Samuel Gross
• 金额: $ 15.29万
• 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/2684964
• 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：格罗斯博士的续签申请旨在解决 转录激活剂如何在体内刺激启动子活性。在 热休克因子(HSF)激活酿酒酵母 至少两个热休克基因--HSP82和HSC82--的转录 减轻核小体对其上游调节区的抑制。 目前尚不清楚这种蛋白质--或任何一种蛋白质--如何介导局部的破坏 启动子染色质结构的展开，是 转录激活。格罗斯博士建议确定这种激活 酵母HSF结构域(YHSF)在体内指导染色质重塑。 首先，使用基因抑制分析，研究人员希望选择 核小体。对这种显性HSF1抑制子的测序应该允许 鉴定天然的重塑结构域；互补的HSF1结构域 将进行删除分析以确认这一分配。第二，Dr。 格罗斯建议对结构和功能表型进行表征 与HSF1突变体相关，并确定它们在两者中的作用 建立和维持靶标温度下的无核小体表型 震撼推进器。第三，解决重塑的机制 结构域工程，申请者将在生物化学上重建HSP82 使用纯化的组分将启动子转化为稳定的二核小体复合体。 将尝试使用HeLa和S.cerevisiae核心进行重建 组蛋白，目标是建立一个完全同源的系统。格罗斯博士将在那时 问重组的yHSF是否可以结合和破坏这个重组的 复杂，无论这一活动是通过存在 SWI/SNF复合体和三磷酸腺苷是否被HSP70抑制 或突变组蛋白，以及功能获得的yHSF突变多肽是否 在体外重塑野生型yHSF方面更有效。最后， 申请人将认为yHSF重塑结构域有能力 与SWI/SNF亚基以及单个核心组蛋白和 一般转录复合体的组成部分，通过执行GST-下拉 实验。这个项目与健康有关的原因是 大量的人类疾病，包括许多癌症，与 启动子特异转录因子的错义突变。 此外，已知SWI/SNF复合体与 增强或抑制人类肿瘤的形成。