EXOGENOUS INDUCTION OF THE E.COLI UHPT GENE

大肠杆菌 UHPT 基因的外源诱导

基本信息

批准号：
6687744
负责人：
ROBERT J KADNER
金额：
$ 25.06万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-07-01 至 2005-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6687744
关键词：
DNA binding protein Escherichia coli bacterial genetics bacterial proteins biological signal transduction chimeric proteins gene induction /repression gene mutation glucose 6 phosphate membrane transport proteins phosphorylation protein protein interaction protein purification protein reconstitution protein structure function site directed mutagenesis

项目摘要

DESCRIPTION (Adapted from applicant's abstract): This project seeks to elucidate how an extracellular signal changes specific gene transcription. The Escherichia coli uhp locus encodes an active transport system, UhpT, for uptake of sugar phosphates and related substrates. Transcription of the uhpT gene is induced by extracellular glucose-6-phosphatte through the action of an unusual two-component regulatory system encoded by the uhpABC genes. Our overall goal is to understand the mechanisms of transmembrane signaling by the UhpBC genes. Complex, the regulation of phosphate transfer to the transcription activator UhpA, the effect of phosphorylation of UhpA on its function, the mechanism of transcription activation by UhpA, and the basis for the co-dependent activation of transcription by UhpA and the global regulatory protein CAP. 1. The segments of UhpB that are involved in recognition of UhpA, and phosphate transfer to and from it, will be probed by genetic and biochemical tests. The role of sequestration of UhpA on the proper control of gene expression will be analyzed. The basis for the inactivity of the isolated kinase segment as auto-kinase will be explored as a function of the exposure of the site of phosphorylation, and the effect of mutations and the presence of UhpC on conformation of the phosphorylation domain. 2. The interactions of the membrane-bound UhpB and UhpC proteins in response to the presence of inducer will be examined by chemical crosslinking studies. The reconstitution of purified full-length UhpB into proteoliposomes, with and without UhpC, will allow biochemical examination of the inducer- specific control of phosphate transfer reactions by UhpB. The role of the hydrophobic N-terminal half of UhpB in membrane anchorage, binding of UhpC, and signal transduction process, by deletion or replacement of individual transmembrane segments. 3. The UhpC protein will be purified and reconstituted into proteoliposomes for assay of inducer binding and transport. Genetic screens will analyze the role of segments of UhpC in inducer binding and specificity. Chimeric proteins containing portions of UhpC and of the transporter UhpT will be made to identify the protein segments that differentiate between the signaling activity of UhpC and the transport activity of UhpT. These studies should provide new information on transmembrane signal transduction coupled between two membrane proteins.

描述（根据申请人的摘要改编）：该项目试图阐明细胞外信号如何改变特定的基因转录。这大肠杆菌UHP基因座编码一个主动运输系统UHPT，以吸收糖磷酸盐和相关底物的UHPT基因的转录是通过不寻常的作用，由细胞外葡萄糖-6-磷酸化诱导由UHPABC基因编码的两个组件调节系统。我们的总体目标是了解UHPBC基因跨膜信号传导的机制。复合物，调节磷酸盐转移到转录激活剂 UHPA，UHPA磷酸化对其功能的影响 UHPA的转录激活和共依赖性激活的基础 UHPA的转录和全球调节蛋白上限。 1。识别UHPA和磷酸盐的UHPB段从遗传和生化测试进行探测，向其转移。这 UHPA的隔离在适当控制基因表达的作用将是分析。分离的激酶段无活动的基础自动激酶将作为现场暴露的函数探索磷酸化以及突变的作用和UHPC的存在磷酸化结构域的构象。 2。响应于膜结合的UHPB和UHPC蛋白的相互作用诱导剂的存在将通过化学交联研究来检查。这与和没有UHPC，将允许对诱导剂的生化检查 UHPB控制磷酸盐转移反应。疏水的作用 UHPB的N末端一半在膜锚固，UHPC的结合和信号的结合通过删除或更换单个跨膜的转导过程细分市场。 3。UHPC蛋白将被纯化并重构为蛋白脂质体诱导者结合和运输的测定。遗传筛选将分析角色 UHPC在诱导剂结合和特异性中的段。嵌合蛋白包含UHPC和运输蛋白UHPT的部分确定区分信号活性的蛋白质段 UHPC和UHPT的运输活动。这些研究应该提供新的关于两个膜之间耦合的跨膜信号转导的信息蛋白质。