TRANSCRIPTION ACTIVATION AT THE RHABAD OPERON

RHABAD 操纵子的转录激活

• 批准号: 6180638
• 负责人: SUSAN M EGAN
• 金额: $ 10.57万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180638
• 关键词: DNA binding protein; DNA directed RNA polymerase; Escherichia coli; bacterial genetics; cytoskeletal proteins; gene expression; gene induction /repression; genetic promoter element; genetic transcription; molecular cloning; mutant; nucleic acid sequence; operon; protein protein interaction; protein sequence; protein structure function; site directed mutagenesis; transcription factor

A model for transcription activation has recently been developed based on the structure of E. Coli RNA polymerase, and analysis of activation by the CRP protein. In this model, the activator protein directly contacts the RNA polymerase alpha or subunit, resulting in increased transcription initiation. The long range goal of this research is to understand the molecular mechanisms of transcription activation, especially where multiple activator proteins simultaneously regulate expression of a gene(s). The objective of this proposal is to investigate the details of transcription activation, utilizing the E. coli L-rhamnose rhaBAD genes as a model. Full transcription of rhaBAD requires two activator proteins, RhaS and CRP. The central hypothesis is that while activator-RNA polymerase interactions likely contribute to transcription activation, activator-activator and activator-DNA interactions may also be important. In order to understand how transcription activation occurs, it is essential to determine the nature of the protein-protein and protein-DNA interactions that are required. Three specific aims will be pursued to accomplish the objectives of this proposal: 1) characterize DNA binding by RhaS; 2) characterize transcription activation by RhaS; and 3) characterize transcription activation by CRP. The role of protein-DNA interactions will be investigated by isolating mutations which identify the amino acid-base contacts between RhaS and DNA. To understand the role of protein-protein interactions in activation, mutations will be isolated that identify the specific amino acids in RhaS and CRP that are responsible for transcription activation. Once activation defective mutations are identified in RhaS and CRP, second site suppressors will be isolated. The second site suppressor mutations will identify the protein-protein interactions that are important for transcription activation. The expectation is that this research will identify a variety of interactions that contribute to transcription activation. This work is significant because it will contribute to a broader understanding of the mechanisms used for activation, especially with multiple activators.

一个转录激活的模型最近被开发出来 根据E.大肠杆菌RNA聚合酶，并分析 由CRP蛋白激活。在这个模型中，激活蛋白 直接接触RNA聚合酶α或亚基，导致 增加转录起始。长期目标是 研究的目的是了解转录的分子机制 激活，特别是当多种激活蛋白 同时调节基因表达。的目标 该提议是研究转录激活的细节， 利用E. coli L-鼠李糖rhaBAD基因作为模型。充分 rhaBAD的转录需要两种激活蛋白RhaS和 CRP.核心假设是，虽然激活RNA 聚合酶相互作用可能有助于转录激活， 活化剂-活化剂和活化剂-DNA相互作用也可以是 重要.为了了解转录激活是如何 发生，它是必不可少的，以确定蛋白质的性质-蛋白质 和蛋白质与DNA的相互作用。 为实现以下目标，将努力实现三个具体目标： 该建议：1）通过RhaS表征DNA结合; 2） 通过RhaS表征转录激活;和3）表征 通过CRP激活转录。蛋白质-DNA的作用 将通过分离突变来研究相互作用， 识别RhaS和DNA之间的氨基酸-碱基接触。到 了解蛋白质-蛋白质相互作用在激活中的作用， 突变将被分离出来，以确定特定的氨基酸， RhaS和CRP负责转录激活。 一旦在RhaS中鉴定出活化缺陷突变， 将分离CRP、第二部位抑制因子。第二站点 抑制基因突变将识别蛋白质-蛋白质相互作用 对转录激活很重要。 人们期望 这项研究将确定各种各样的相互作用， 有助于转录激活。这项工作意义重大 因为它将有助于更广泛地理解 用于激活的机制，特别是使用多个激活剂。