MECHANISMS OF YEAST TRANSCRIPTIONAL INITITATION

酵母转录起始机制

• 批准号: 2175717
• 负责人: KEVIN STRUHL
• 金额: $ 56.46万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-03-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175717
• 关键词: DNA binding protein; DNA directed RNA polymerase; Saccharomyces; X ray crystallography; affinity chromatography; chromatin; chromosome aberrations; fungal genetics; gene deletion mutation; gene expression; genetic library; genetic manipulation; genetic mapping; genetic promoter element; genetic recombination; genetic transcription; molecular cloning; mutant; nucleic acid sequence; point mutation; protein engineering; protein sequence; recombinant DNA; regulatory gene; structural genes; suppressor mutations; temperature sensitive mutant; transcription factor

The mechanisms by which eukaryotic organisms regulate gene expression are important for basic scientific knowledge that is necessary for understanding many complex biological phenomena including human diseases. With regard to the process of transcriptional initiation, a wide variety of experiments have pointed to common molecular mechanisms in eukaryotic organisms ranging from humans to yeasts. This proposal will continue to investigate several basic issues concerning the molecular mechanisms of transcriptional regulation in yeast by combining a wide variety of techniques including recombinant DNA technology, molecular and classical yeast genetics, and protein and nucleic acid biochemistry. First, we will investigate the various functions of the TFIID by obtaining mutants with the following properties: support basal transcription but fail to respond to acidic activator proteins; are specifically defective either for transcription by RNA polymerase II or RNA polymerase III; show differential effects at TATA-containing vs TATA-less promoters; fail to support cell growth but retain RNA polymerase II function. The resulting mutants will be characterized biochemically for TATA-binding activity, interactions with TFIIA, TFIIB (and potentially other proteins), and by in vitro transcription. Second, we will define gene products, either by suppressor mutations or by overexpression, that revert phenotypes conferred by defective TFIID derivatives. We will use the "two-hybrid" method for detecting protein-protein interactions to search (as well as test) for TFIID-interacting proteins and to map the interacting surfaces. The genes and the encoded proteins will be characterized by standard techniques of yeast molecular biology. Third, having isolated recessive and dominant suppressor mutations that increase transcription by GCN4 derivatives with partially defective acidic activation regions, we will carry out detailed allele specificity experiments and clone the encoding genes. The goals are to identify proteins that are involved either in the mechanism by which acidic activation domains stimulate the basic transcription machinery or in regulation of GCN4 activity. Fourth, to understand the biological functions of the yeast ATF/CREB protein family, we will perform a mutational analysis of ACR1, identify and characterize ATF/CREB activators, and develop a novel method to identify target genes that are directly regulated by the various ATF/CREB proteins in vivo. Fifth, to determine the mechanisms underlying the functional distinctions between the his3 TATA elements, we will investigate the effect of different activator proteins, the quality of the acidic activation domain, the length of the poly(dA).poly(dT) element and other sequences that affect chromatin structure, competition between TATA elements, and mutations in already identified genes that affect transcription. In addition, we will isolate chromosomal mutations that cause differential effects on transcription mediated by these TATA elements. Sixth, the biochemical functions of the various proteins and protein variants will be examined by in vitro transcription on appropriate promoters using yeast nuclear extracts from wild-type or mutant strains, extracts that have been depleted for TFIID (or other factors), and purified general transcription factors. In addition, protein-protein interaction studies will be carried out by employing standard band-shift assays and affinity chromatography. Seventh, we will obtain new GCN4 derivatives that have altered DNA-binding specificity and design GCN4 mutants and target sites to test specific hypotheses that arise from the X-ray structure of the protein-DNA complex.

真核生物调节基因表达的机制是 重要的基础科学知识，是必要的， 了解包括人类疾病在内的许多复杂的生物现象。 关于转录起始的过程， 的实验已经指出了真核生物中常见的分子机制， 从人类到酵母菌。 该提案将继续 研究有关分子机制的几个基本问题， 酵母中的转录调控，通过结合各种各样的 包括重组DNA技术、分子和经典的 酵母遗传学，蛋白质和核酸生物化学。 一是 将通过获得突变体来研究TFIID的各种功能 具有以下特性：支持基础转录，但不能 对酸性激活蛋白有反应;有特异性缺陷， 用于通过RNA聚合酶II或RNA聚合酶III转录;显示 在含TATA的启动子与不含TATA的启动子上的差异效应;未能 支持细胞生长但保留RNA聚合酶II功能。 所得 突变体的TATA结合活性将被生物化学表征， 与TFIIA、TFIIB（以及潜在的其他蛋白质）的相互作用，以及 体外转录 第二，我们将定义基因产物， 抑制基因突变或过度表达， 由有缺陷的TFIID衍生物赋予。 我们将用“两混” 用于检测蛋白质-蛋白质相互作用以搜索的方法（以及 测试）的TFIID相互作用的蛋白质和映射相互作用的表面。 基因和编码的蛋白质将通过标准方法表征。 酵母分子生物学技术。 第三，具有孤立隐性 和显性抑制基因突变， 具有部分缺陷的酸性活化区域的衍生物，我们将 进行详细的等位基因特异性实验， 基因. 目标是鉴定参与以下两种蛋白质： 酸性激活结构域刺激碱性 转录机制或调节GCN 4活性。 四是大力 了解酵母ATF/CREB蛋白家族的生物学功能， 我们将对ACR 1进行突变分析， ATF/CREB激活剂的研究进展，并建立了一种新的靶基因鉴定方法 在体内由各种ATF/CREB蛋白直接调节。 第五，确定职能区别的机制 在his 3 TATA元件之间，我们将研究 不同的激活蛋白，酸性激活的质量 结构域、poly（dA）.poly（dT）元件和其它序列的长度 影响染色质结构、TATA元件之间的竞争，以及 影响转录的基因突变。 在 此外，我们将分离染色体突变，导致差异 对这些TATA元件介导的转录的影响。 六是 各种蛋白质和蛋白质变体的生物化学功能将 通过在合适的启动子上的体外转录进行检测， 来自野生型或突变株的酵母核提取物， 已耗尽TFIID（或其他因素），并纯化一般 转录因子 此外，蛋白质-蛋白质相互作用研究 将通过使用标准带移测定和亲和层析来进行 层析 第七，我们将获得新的GCN 4衍生物， 改变DNA结合特异性和设计GCN 4突变体和靶位点 为了测试由X射线结构产生的特定假设， 蛋白质-DNA复合物