RNA POLYMERASE SIGMA SUBUNIT--STRUCTURE AND FUNCTION

描述：本申请建议扩展E. 大肠杆菌RNA聚合酶主要sigma亚基（sigma-70），sigma-70 与核心聚合酶亚基β引物，和各种竞争 σ家族的成员用于结合到核心。具体而言，目标是： 研究核心聚合酶亚基β prime与sigma-70的相互作用。 我们最近开发了一种强大的新方法（有序片段梯 远Western分析）用于蛋白质-蛋白质相互作用结构域的作图， 我用它来定位sigma-70与氨基酸260 - 309的主要结合位点 beta prime subunit的缩写。我们会继续进行这些研究， 这种相互作用的确切性质β撇260 - 309区域的β 主亚基我们将继续进行诱变研究， 在存在和不存在σ-70的情况下，通过NMR测定β撇260 - 309。我们将 确定在sigma-70上发生这种相互作用的位置，并测试 假设其他σ家族成员的同源区域与 在贝塔质数上的同一个网站。 研究sigma家族成员对核心蛋白的竞争。我们生产过剩， 纯化了所有7个E。大肠杆菌sigmas，并开发了免疫试剂， 方法来准确地量化它们。我们将测量结合参数 在几种离子条件下的每个σ的核心。我们会研究 这些不同σ对体外纯化的核心聚合酶的竞争 系统.我们将使用我们的绑定结果来模拟多重交互 在sigma和core之间。我们将通过诱导适度的 然后确定这种扰动对 使用DNA在七个sigma中的每一个的控制下表达基因， 微阵列通过这种方式，我们将测试的假设，全球监管的 转录是由σ竞争核心决定的。 探索我们研究结果的医学意义。的详细知识 sigma-70和其他sigma与核心相互作用具有潜在的医疗 应用.我们学习的一个长期目标是， 特定的小分子会干扰这些关键的相互作用。我们 将识别结合和干扰的肽和/或小分子， σ-核心结合。这可能会导致一个新的类别的发展， 抗生素

DESCRIPTION: This application proposes to extend detailed studies of the E. coli RNA polymerase major sigma subunit (sigma-70), the interaction of sigma-70 with the core polymerase subunit beta prime, and the competition of various members of the sigma family for binding to core. Specifically, the aims are: To study the interaction of core polymerase subunit beta prime with sigma-70. We have recently developed a powerful new method (ordered fragment ladder far-Western analysis) for mapping protein-protein interaction domains and have used it to map a major binding site for sigma-70 to the amino acid 260-309 region of the beta prime subunit. We will continue these studies to determine the precise nature of this interaction of beta prime 260-309 region of the beta prime subunit. We will continue mutagenesis studies and determine the structure of beta prime 260-309 by NMR in the presence and absence of sigma-70. We will determine where on sigma-70 this interaction is occurring and test the hypothesis that homologous regions of other sigma family members interact with this same site on beta prime. To study competition of sigma-family members for core. We have overproduced and purified all seven E. coli sigmas and have developed immunological reagents and methods to accurately quantitate them. We will measure the binding parameters of each sigma for core under several ionic conditions. We will study the competition of these various sigmas for core polymerase in purified in vitro systems. We will use our binding results to model the multiple interactions among sigmas and core. We will perturb in vivo conditions by inducing moderate overproduction of a sigma and then determine the effect of this perturbation on the expression of genes under the control of each of the seven sigmas using DNA microarrays. In this way we will test the hypothesis that global regulation of transcription is determined by sigma competition for core. To explore the medical implications of our results. Detailed knowledge of the interactions of sigma-70 and other sigmas with core has potential medical applications. A long-term goal of our studies is to learn enough to devise specific small molecules that will interfere with these key interactions. We will identify peptides and/or small molecules that bind to and interfere with sigma-core binding. This might lead to the development of a new class of antibiotics.