RNA POLYMERASE SIGMA SUBUNIT--STRUCTURE AND FUNCTION

描述：本申请建议扩展对E。 大肠杆菌RNA聚合酶主要Sigma亚基(Sigma-70)，Sigma-70的相互作用 与核心聚合酶亚基贝塔质数，以及各种 西格玛家族的成员，用于与核心结合。具体来说，目标是： 研究核心聚合酶β亚基与Sigma-70的相互作用。 我们最近开发了一种强大的新方法(有序片段梯形图 Far-Western分析)来绘制蛋白质-蛋白质相互作用结构域，并具有 利用它将Sigma-70的一个主要结合位点定位到260-309氨基酸 贝塔质亚单位的区域。我们将继续进行这些研究，以确定 贝塔质数260-309区域相互作用的精确性质 质子亚单位。我们将继续进行诱变研究并确定其结构 在Sigma-70存在和不存在的情况下，通过核磁共振测定β素数260-309。我们会 确定Sigma-70上发生此交互的位置，并测试 其他西格玛家族成员的同源区域相互作用的假设 这是同一个网站的测试版。 研究西格玛家族成员对核心的竞争。我们已经生产过剩， 纯化了所有七种大肠杆菌Sigma，并开发了免疫试剂和 方法准确定量。我们将测量绑定参数 在几种离子条件下，岩心的每一西格玛。我们会研究 不同类型Sigma在体外纯化中对核心聚合酶的竞争 系统。我们将使用绑定结果对多个交互进行建模 在西格玛和核心之间。我们将通过诱导适度的诱导来扰乱体内条件 Sigma的生产过剩，然后确定这种扰动对 用DNA控制七个西格玛基因的表达 微阵列。通过这种方式，我们将检验这样一个假设，即全球监管 转录是由核心的西格玛竞争决定的。 来探索我们研究结果的医学意义。详细了解 Sigma-70和其他Sigma与核心的相互作用具有潜在的医学意义 申请。我们研究的一个长期目标是学习足够多的东西来设计 会干扰这些关键相互作用的特定小分子。我们 将识别与其结合并干扰的多肽和/或小分子 西格玛-核心绑定。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.