STRUCTURE AND REGULATION OF THE YEAST HSP90 GENES

酵母 HSP90 基因的结构和调控

• 批准号: 3305294
• 负责人: David Samuel Gross
• 金额: $ 9.87万
• 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/3305294
• 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， 已知的系统;它是所有生命，原核生物和 真核生物 它代表了细胞 承受生理压力，无论是热、化学或缺氧， 因此可能在发烧期间细胞存活中起关键作用， 乙醇中毒和局部缺血。 几乎无一例外， 应答在转录水平上协调调节。 我们希望了解这种转录的分子基础 调节：什么激活热休克基因下非诱导 （基础）条件下，是什么原因导致他们的感应反应， 环境压力，以及什么限制了他们的反应， 持续的压力。 为了解决这些问题，我们建议 以酿酒酵母的HSP 90基因家族为模型， 系统 这个家庭由两个成员组成， 在它们的调节中是惊人的，但是它们编码一种功能性的 无法区分的基因产物 HSP 82在低基础水平表达， 热休克可使HSC 82水平提高10至20倍; HSC 82是 表达水平比HSP 82高10倍，但 是由压力诱导的两倍。 的具体问题 我们建议处理以下问题。 第一，什么是顺- 调控元件激活HSP 82？ 我们希望执行一个 基因启动子区的系统性5 '缺失分析， 采用阿托伐他汀定向诱变和基因 置换技术，引入位点特异性突变， 两个序列元件--TATA盒和启动子近端热 冲击元件，HSE 1--我们先前的工作已经表明， 在体内参与蛋白质/DNA相互作用。 功能 将通过北方印迹分析评估结果; 结果通过染色质足迹使用化学和酶 探针在核小体和核苷酸分辨率。 第二、 哪些顺式调节元件负责10倍的 HSC 82的基础表达水平高于HSP 82？ 解决 这个问题，我们建议进行互补诱变 和HSC 82启动子的足迹分析。 第三， 反式作用蛋白激活这些热休克基因， 负责限制慢性期的反应， 压力？ 使用酵母热休克因子（HSF）的抗体，酵母 TATA结合因子，TFIID，酵母RNA的最大亚基 聚合酶II和酵母hsp 70（ssa 1 p），我们建议 免疫沉淀共价交联蛋白质/DNA复合物 从热休克和对照细胞中纯化，并通过印迹鉴定- 杂交那些DNA序列与每个 体内蛋白质