STRUCTURE AND REGULATION OF THE YEAST HSP90 GENES

热休克反应是高度保守的基因之一。 已知的系统；它对所有生命、原核生物和 真核生物。它表示单元格使用的主要方法 耐受生理压力，无论是热、化学或缺氧，以及 因此可能对细胞在发烧期间的存活起着关键作用， 酒精中毒和脑缺血。几乎无一例外地， 反应是在转录水平上协调调节的。 我们希望了解这种转录的分子基础。 调控：在非诱导条件下是什么激活了热休克基因 (基础)条件，是什么导致它们对 环境压力，以及是什么限制了它们在经期的反应 持续的压力。为解决这些问题，我们建议 以酿酒酵母HSP90基因家族为模型 系统。该家族由两个不同的成员组成 令人震惊的是在他们的规则中，但在功能上编码 难以辨别的基因产物。HSP82在基础水平较低时表达 通过热冲击提高10到20倍的水平；HSC82是 在比HSP82高10倍的组成水平上表达，但 在压力的作用下只会进一步增加2倍。具体问题 我们建议解决以下问题。首先，哪个顺- 调控元件激活HSP82？我们希望执行一项 对该基因启动子区域进行系统的5‘-缺失分析， 利用寡核苷酸定向突变和基因 移位技术，将定点突变引入到 两个序列元件--TATA盒和启动子--近端热 令人震惊的元素，HSE1--我们之前的工作表明， 在体内参与蛋白质/DNA的相互作用。功能性 后果将通过Northern印迹分析进行评估；结构性 使用化学和酶的染色质足迹的后果 核小体和核苷酸分辨率的探针。第二, 哪些顺式监管因素导致了10倍的增长 HSC82的基础表达水平高于HSP82？致信地址 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，哪一个 反式作用蛋白激活这些热休克基因，而 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 TATA结合因子，TFIID，酵母RNA最大亚基 聚合酶II和酵母热休克蛋白70(Ssa1p)，我们建议 免疫共沉淀法制备共价交联的蛋白质/DNA复合物 从热休克细胞和对照细胞中纯化，并通过印迹鉴定- 将这些密切接触的DNA序列进行杂交 体内的蛋白质。

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.