STRUCTURE AND REGULATION OF THE YEAST HSP90 GENES

酵母 HSP90 基因的结构和调控

• 批准号: 3305295
• 负责人: David Samuel Gross
• 金额: $ 10.64万
• 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 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3305295
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA footprinting; Saccharomyces cerevisiae; environmental stressor; fungal genetics; gene deletion mutation; gene expression; genetic promoter element; genetic regulatory element; genetic transcription; immunoprecipitation; northern blottings; nucleic acid sequence; point mutation; site directed mutagenesis; stress proteins; transcription factor

The heat shock response is among the most highly conserved genetic systems known; it is essential for all life, prokaryotic and eukaryotic. It represents the principal means by which cells endure physiologic stress, be it thermal, chemical, or anoxic, and thus likely plays a key role in cell surviving during fever, ethanol toxicity, and ischemia. Almost without exception, the response is coordinately regulated at the level of transcription. We wish to understand the molecular basis of this transcriptional regulation: what activates heat shock genes under non-inducing (basal) conditions, what causes their induction in response to environmental stress, and what limits their response during periods of continuous stress. To address these questions, we propose to use the HSP90 gene family of Saccharomyces cerevisiae as a model system. This family consists of two members which differ strikingly in their regulation but which encode a functionally indistinguishable gene product. HSP82 is expressed at a low basal level which is enhanced 10-to-20-fold by heat shock; HSC82 is expressed at a 10-fold higher constitutive level than is HSP82, but is induced only 2-fold further by stress. The specific questions we propose to address are the following. First, which cis- regulatory elements activate HSP82? We wish to perform a systematic 5'-deletion analysis of the gene's promoter region and, employing oligonucleotide-directed mutagenesis and gene transplacement techniques, introduce site-specific mutations into two sequence elements--the TATA box and the promoter-proximal heat shock element, HSE1--our prior work has shown are intimately engaged in protein/DNA interactions in vivo. Functional consequences will be assessed by Northern blot analysis; structural consequences by chromatin footprinting using chemical and enzymatic probes at both nucleosome- and nucleotide-resolution. Second, which cis-regulatory elements are responsible for the 10-fold higher basal level of expression of HSC82 vs. HSP82? To address this question, we propose to perform a complementary mutagenesis and footprinting analysis of the HSC82 promoter. Third, which trans-acting proteins activate these heat shock genes, and which are responsible for limiting the response during periods of chronic stress? Using antibodies to yeast heat shock factor (HSF), yeast TATA-binding factor, TFIID, the largest subunit of yeast RNA polymerase II, and yeast hsp70 (ssa1p), we propose to immunoprecipitate covalently crosslinked protein/DNA complexes purified from heat shocked and control cells, and identify by blot- hybridization those DNA sequences in intimate contact with each protein in vivo.

热休克反应是最高度保守的遗传反应之一 系统已知；它对于所有生命（原核生物和生物）都是必需的 真核的。 它代表了细胞的主要手段 承受生理压力，无论是热、化学还是缺氧，并且 因此可能在发烧期间细胞的存活中发挥关键作用， 乙醇毒性和缺血。 几乎无一例外的是， 反应在转录水平上得到协调调节。 我们希望了解这种转录的分子基础 调节：非诱导下什么激活热休克基因 （基础）条件，是什么导致它们响应 环境压力以及哪些因素限制了他们在月经期间的反应 持续的压力。 为了解决这些问题，我们建议 使用酿酒酵母的HSP90基因家族作为模型 系统。 这个家庭由两个不同的成员组成 它们的调节引人注目，但编码了功能 无法区分的基因产物。 HSP82 的基础表达水平较低 热激后的水平可提高10至20倍； HSC82 是 其表达水平比 HSP82 高 10 倍，但是 压力仅进一步诱导 2 倍。 具体问题 我们建议解决以下问题。 首先，哪个顺式 调控元件激活HSP82？ 我们希望执行一个 对基因启动子区域进行系统的 5' 缺失分析， 采用寡核苷酸定向诱变和基因 置换技术，将位点特异性突变引入 两个序列元件——TATA盒和启动子-近端热 冲击元件，HSE1——我们之前的工作已经表明它们是密切相关的 参与体内蛋白质/DNA相互作用。 功能性 后果将通过 Northern blot 分析进行评估；结构性的 使用化学和酶法进行染色质足迹的后果 核小体和核苷酸分辨率的探针。 第二， 哪些顺式调控元件负责 10 倍 HSC82 的基础表达水平高于 HSP82？ 致地址 这个问题，我们建议进行互补诱变 以及HSC82启动子的足迹分析。 第三，哪个 反式作用蛋白激活这些热休克基因，并且 负责限制慢性病期间的反应 压力？ 使用酵母热休克因子 (HSF) 抗体，酵母 TATA 结合因子，TFIID，酵母 RNA 的最大亚基 聚合酶 II 和酵母 hsp70 (ssa1p)，我们建议 免疫沉淀共价交联的蛋白质/DNA 复合物 从热休克细胞和对照细胞中纯化，并通过印迹法进行鉴定 杂交那些与每个密切接触的DNA序列 体内的蛋白质。