REGULATION AND FUNCTION OF FIS IN E COLI

FIS 在大肠杆菌中的调节和功能

• 批准号: 2634774
• 负责人: ROBERT OSUNA
• 金额: $ 10.32万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2634774
• 关键词: DNA binding protein; Escherichia coli; bacterial proteins; gel electrophoresis; gel mobility shift assay; gene deletion mutation; genetic regulation; molecular cloning; nucleic acid sequence; operon; point mutation; protein structure function; ribosomes; site directed mutagenesis; southern blotting; transfer RNA; transposon /insertion element

The ability of cells to efficiently respond to changes in environmental signals can be of fundamental importance in their function and survival. Such processes often depend on rapid expression of cellular factors that can signal a proper and timely response. In Escherichia coli, the small DNA binding protein Fis is involved in an increasing number of cellular processes that include stimulation of site-specific DNA recombination, stimulation of ribosomal and tRNA operon transcription, and initiation of DNA replication at oriC. The expression pattern of Fis is dramatic and unprecedented for a bacterial protein. Although Fis is not detected during stationary phase, a rapid but transient increase to 50,000-100,000 Fis molecules per cell succeeds a nutrient upshift. Its activation at the mRNA level is not dependent on de novo protein synthesis. In these respects, fis is analogous to the eukaryotic primary response genes whose mRNA synthesis is induced by external signals without the need for newly synthesized proteins, and whose products often mediate a cellular response. A number of primary response genes have been shown to be protooncogenes (i.e. myc,jun, and fos) and, as such, are implicated in growth control. The potential role of Fis as a global regulator of a number of cellular processes, some of which are crucial for attaining optimal cell growth conditions, makes Fis an excellent candidate for a mediating factor between external signals in the medium and a rapid cellular response. Our long term objective is to understand the physiological significance of the novel fis regulation pattern as well as the molecular properties that allow this protein to execute widely different biological functions. In this work we wish to ask: how is fis regulated?, what other cellular functions are regulated by Fis? and, how does Fis mediate its effects? We will identify the trans and cis acting elements responsible for the unusual fis regulation pattern. Gel mobility shift assays of crude extracts will be used to identify fis promoter-specific binding proteins. Transposition mutagenesis will also be performed to identify host genes whose products are required for fis regulation. Cis acting regulatory sites will be identified using a combination of DNA deletion and point mutation analysis of the fis promoter region. We will use in vivo footprinting techniques to identify regulatory sites that are differentially occupied during activation and repression of fis. Turbidistat culturing conditions will be used to examine the effects of different growth media on fis expression. Novel regulator functions of Fis will be identified using two dimensional gel electrophoresis of total proteins synthesized in the presence or absence of Fis at various stages of growth. Other approaches to identify Fis-regulated genes will make use of a) a hybridization method in which of a large collection of E. coli clones are probed with 32P-labeled cDNA made from total RNA synthesized in the presence or absence of Fis, and b) transposition of lambda-plac-Mu on the E. coli chromosome to select lacZ fusions to fis-regulated promoters. Finally, we will gene rate random and targeted mutations in fis to study the molecular basis by which Fis recognizes different DNA sites and interacts with other proteins to promote diverse cellular processes.

细胞有效响应环境变化的能力 信号在其功能和生存中可能至关重要。 这样的过程通常取决于细胞因素的快速表达 可以发出适当及时的响应。在大肠杆菌中 DNA结合蛋白FIS参与越来越多的细胞 包括刺激位点特异性DNA重组的过程， 刺激核糖体和tRNA操纵子转录，并开始 DNA复制在oric。 FI的表达模式是戏剧性的，并且 用于细菌蛋白的前所未有。虽然在 固定阶段，快速但短暂的增加到50,000-100,000 fis 每个细胞分子成功地降低了营养。它在mRNA上的激活 水平不取决于从头蛋白的合成。在这些方面， FIS类似于真核原代反应基因的mRNA 综合是由外部信号诱导的，而无需新的 合成的蛋白质，其产物经常介导细胞 回复。许多主要响应基因已显示为 原子基因（即myc，jun和fos），因此与 增长控制。 FI作为全球监管者的潜在作用 细胞过程数量，其中一些对于获得至关重要 最佳细胞生长条件使FIS成为A 介质中外部信号之间的中介因子和快速 细胞反应。 我们的长期目标是了解 新型FIS调节模式以及分子特性 允许该蛋白质执行广泛不同的生物学功能。在 我们希望问的这项工作：FIS如何受到调节？，其他哪些蜂窝 功能受FIS的调节？而且，FIS如何介导其效果？我们 将确定负责 不寻常的FIS调节模式。凝胶机动性转移测定法 提取物将用于鉴定FIS启动子特异性结合蛋白。 还将进行换位诱变以识别宿主基因 FIS调节需要其产品。顺式代理监管 将使用DNA缺失和点的组合来识别站点 FIS启动子区域的突变分析。我们将使用体内 识别监管站点的足迹技术 在FIS激活和抑制过程中占据差异。 浑浊的培养条件将用于检查 关于FIS表达的不同增长培养基。 FIS的新型调节函数 将使用总计的二维凝胶电泳来识别 在不同阶段存在或不存在FIS的情况下合成的蛋白质 增长。识别FIS调节基因的其他方法将使用 a）一种杂交方法，其中大量大肠杆菌集 用由由总RNA制成的32p标记的cDNA探测克隆 FIS的存在或不存在，b）lambda-plac-mu的换位 大肠杆菌染色体选择与FIS调节的启动子选择LACZ融合。 最后，我们将在FIS中随机基因率和靶向突变进行研究 FIS识别不同DNA位点的分子基础和 与其他蛋白质相互作用以促进各种细胞过程。