EXOGENOUS INDUCTION OF THE E. COLI UHPT GENE

大肠杆菌 UHPT 基因的外源诱导

• 批准号: 3295290
• 负责人: ROBERT J KADNER
• 金额: $ 12.82万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295290
• 关键词: DNA binding protein; DNA footprinting; Escherichia coli; active transport; alkaline phosphatase; autoradiography; bacterial genetics; beta galactosidase; binding proteins; bioenergetics; chromosome deletion; cyclic AMP receptors; density gradient ultracentrifugation; enzyme mechanism; gel electrophoresis; gene expression; gene induction /repression; gene interaction; gene mutation; genetic mapping; genetic promoter element; genetic transcription; genetic translation; laboratory mouse; laboratory rabbit; molecular cloning; molecular genetics; nucleic acid hybridization; nucleic acid sequence; point mutation; regulatory gene; site directed mutagenesis; structural genes

Transcription of the Escherichia coli uhpT gene, which encodes an active transport system for sugar phosphates, is induced by extracellular, but not by intracellularly generated glucose-6- phosphate. The uhpT gene and the three contiguous regulatory genes have been cloned and their nucleotide sequence determined. All three regulatory genes are required for proper regulation. UhpA appears to be an activator of uhpT transcription, whereas UhpB and UhpC appear to act to prevent UhpA action in the absence of external inducer. Projects in this proposal address the regulation of this system through the isolation of the three regulatory proteins, and the exploration of their interactions with inducer, the membrane, each other, and the uhpT promoter region. Both biochemical and genetic approaches will be employed to investigate these interactions. Deletion and point mutations in the uhpT promoter region will be combined with footprinting analyses to determine the sites of binding of UhpA, RNA polymerase, and the cAMP receptor protein. Mutants that overcome the loss of one regulatory protein will be isolated in hopes that they will provide further information on the interaction of the regulatory components. Ultimately, attempts will be made to establish an in vitro transcription-translation system that reproduces proper regulation. Since the UhpT transporter has recently been shown to number function as a phosphate antiport, point mutations and fusions to the structural genes for alkaline phosphatase and beta-galactosidase will be isolated, with the goal of defining the transmembrane orientation of the protein. Studies will be addressed to the use of site- directed mutagenesis for identification of residues involved in transport function, substrate recognition, or energy coupling. It is anticipated that these studies will provide information about both the novel regulatory system and its transmembrane signalling, as well as about this novel mechanism of energy coupling.

大肠杆菌UHPT基因的转录，该基因编码 糖磷酸盐的主动运输系统由 细胞外，但不是细胞内产生的葡萄糖-6- 磷酸盐。 UHPT基因和三个连续调节 基因已被克隆，并确定其核苷酸序列。 适当调控所需的所有三个调节基因均需要。 UHPA似乎是UHPT转录的激活剂，而 UHPB和UHPC似乎采取行动防止UHPA动作 没有外部诱导剂。 此提案中的项目解决 通过隔离三个来调节该系统 调节蛋白及其与他们与之相互作用的探索 诱导者，膜，彼此和UHPT启动子 地区。 生化方法和遗传学方法都将是 用于研究这些相互作用。 删除和点 UHPT启动子区域中的突变将与 足迹分析以确定UHPA结合位点， RNA聚合酶和CAMP受体蛋白。 突变体 克服一种调节蛋白的损失将在 希望他们能提供有关 调节组件的相互作用。 最终，尝试 将建立体外转录翻译 重现适当调节的系统。 自UHPT以来 最近已显示转运蛋白作为一个数字函数 磷酸盐反物质，点突变和融合到结构 碱性磷酸酶和β-半乳糖苷酶的基因将是 孤立，目的是定义跨膜方向 蛋白质。 研究将用于使用站点 - 定向诱变，以鉴定涉及的残基 传输功能，底物识别或能量耦合。 这是 预计这些研究将提供有关两者的信息 新型的调节系统及其跨膜信号传导，如 以及这种新颖的能量耦合机制。