EXOGENOUS INDUCTION OF THE E COLI UHPT GENE

大肠杆菌 UHPT 基因的外源诱导

• 批准号: 2734575
• 负责人: ROBERT J KADNER
• 金额: $ 19.52万
• 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 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734575
• 关键词: DNA binding protein; DNA directed RNA polymerase; Escherichia coli; bacterial genetics; bacterial proteins; biological signal transduction; chimeric proteins; gene induction /repression; gene mutation; genetic promoter element; glucose 6 phosphate; intermolecular interaction; membrane transport proteins; phosphorylation; polymerase chain reaction; protein kinase; protein sequence; protein structure function; site directed mutagenesis; stoichiometry

DESCRIPTION: This project seeks to elucidate the mechanisms by which a signal outside the cell leads directly to changes in transcription activity at a gene. The Escherichia coli uhp locus encodes an active transport system UhpT for the uptake of a variety of sugar phosphates and related organophosphates. Transcription of the uhpT gene is induced by the presence of extracellular glucose 6-phosphate (Glu6P), but not by internally generated Glu6P, through the action of three regulatory proteins encoded by the upstream uhpABC locus. Our goal is to understand in molecular detail the mechanism of transmembrane regulation of uhpT transcription by a phospho-relay cascade. The UhpA and UhpB proteins are members of an underexplored subfamily of the widespread group of bacterial two-component regulatory proteins, and have several unusual features. The UhpC protein is involved in transmembrane signaling but is related in structure and sequence to the UhpT family of transport proteins. The specific aims for the requested period of support fall into four areas. 1. The DNA sites for binding of UhpA and the global regulator CAP will be defined, in terms of their sequence determinants, stoichiometry and cooperative of protein binding, and interaction with RNA polymerase. 2. The portions of the UhpA protein that participate in determining DNA binding and transcription activation will be determined by genetic techniques, and the effect of the phosphorylation of UhpA on its oligomeric state, DNA binding affinity, and effect on transcription will be studied through biochemical approaches. 3. The portions of UhpB that are involved in auto-phosphorylation, transfer of phosphate to UhpA, and stimulation of phosphate release from UhpA will be probed by directed mutagenesis. 4. The interactions of the membrane-embedded UhpB and UhpC proteins that comprise the transmembrane signaling complex will be determined by isolation of allele-specific suppressors of mutants with altered regulation, by affinity chromatography, and by reconstitution of the regulatory system in membrane vesicles. Chimeric proteins combining portions of UhpC and the UhpT transport protein will attempt to identify portions of these homologous proteins that contribute to transport and signaling functions, and to substrate specificity. It is anticipated that completion of the above aims will provide deeper insight into the action of an unusual regulatory system, but one which operates in essence in the control of many bacterial virulence factors.

描述：该项目旨在阐明一种机制 电池之外的信号直接导致转录活性的变化 在基因。 大肠杆菌UHP基因座编码主动传输 系统UHPT用于吸收各种糖磷酸盐和相关的系统 有机磷酸盐。 UHPT基因的转录是由存在诱导的 细胞外葡萄糖6-磷酸（GLU6P），但不通过内部 通过三种由三种调节蛋白编码的作用产生的GLU6P 上游UHPABC基因座。 我们的目标是了解分子细节 UHPT转录的跨膜调节机制 Phose-Relay级联。 UHPA和UHPB蛋白是 广泛的细菌两组分子的未置换的亚家族 调节性蛋白质，并具有几个异常特征。 UHPC蛋白是 参与跨膜信号传导，但在结构和序列上相关 到UHPT运输蛋白家族。 具体目标 要求的支持期分为四个领域。 1。 将根据UHPA和全球监管机上限的约束力定义 它们的序列决定因素，化学计量和蛋白质合作 结合，并与RNA聚合酶相互作用。 2。UHPA的部分 参与确定DNA结合和转录的蛋白质 激活将由遗传技术确定，以及 UHPA在其低聚状态，DNA结合亲和力和 将通过生化方法研究对转录的影响。 3。 UHPB的一部分参与自动磷酸化，转移 磷酸盐至UHPA，刺激从UHPA释放的磷酸盐将是 通过定向诱变进行探测。 4。 膜包含膜的UHPB和UHPC蛋白，该蛋白包括跨膜 信号传导复合物将通过分离等位基因特异性来确定 通过亲和力色谱法改变了调节变化的突变体的抑制剂， 并通过重建膜囊泡中的调节系统。 嵌合蛋白结合了UHPC的部分和UHPT转运蛋白 将尝试确定这些同源蛋白的一部分 有助于运输和信号功能，并为底物做出贡献 特异性。 预计上述目标的完成将会 提供对异常监管系统的作用的更深入了解，但 一个在许多细菌毒力控制中本质上运作的 因素。