TRANSCRIPTION ACTIVATION AT THE RHABAD OPERON

最近开发了一种转录激活模型 根据E.Coli RNA聚合酶的结构，分析 被C反应蛋白激活。在这个模型中，激活蛋白 直接接触RNA聚合酶α或亚单位，导致 转录启动增加。这样做的长期目标是 研究是为了了解转录的分子机制 激活，特别是当多个激活蛋白 同时调节一个基因的表达(S)。的目标是 这项提议是为了研究转录激活的细节， 以大肠杆菌L-鼠李糖rhaBAD基因为模型。饱满 RhaBAD的转录需要两种激活蛋白，rHAS和rHAS C反应蛋白。中心假说是，虽然激活剂-RNA 聚合酶相互作用可能有助于转录激活， 激活剂-激活剂和激活剂-DNA相互作用也可以是 很重要。为了理解转录激活是如何 发生时，必须确定蛋白质的性质--蛋白质 以及所需的蛋白质-DNA相互作用。 将追求三个具体目标，以实现以下目标 该建议：1)表征RHAS结合的DNA；2) 通过rHas表征转录激活；以及3)表征 转录被C反应蛋白激活。蛋白质-DNA的作用 相互作用将通过分离突变来研究 鉴定RHAs和DNA之间的氨基酸-碱基接触。至 了解蛋白质相互作用在激活中的作用， 将分离出识别特定氨基酸的突变 负责转录激活的RHAs和CRP。 一旦在RHAS和RHAS中发现激活缺陷突变 C反应蛋白，第二个位点抑制子将被隔离。第二个站点 抑制子突变将识别蛋白质之间的相互作用 它们对转录激活很重要。人们的期望是 这项研究将确定各种相互作用， 有助于转录激活。这项工作意义重大 因为这将有助于更广泛地理解 用于激活的机制，尤指具有多个激活剂的。

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.