Transcription termination and gene regulation by Rho: Integrative analysis

项目摘要/摘要 好了！ 本项目旨在阐明终止因子Rho在细菌细胞中作用的未知方面。 我们发现，Rho是比预期大得多的监管网络的一部分，包括 核糖开关和sRNA。我们还发现了许多由Rho形成的蛋白质-蛋白质相互作用， 不依赖于核酸，允许其对全球基因表达的影响由上游调控 监管信号。在这份提案中，我们通过部署多方面的集成来建立在这些发现的基础上 侧重于Rho相互作用和细胞内非正则函数的分析工具。首先，我们将在体内使用 发现Rho形成的蛋白质复合体的结构相互作用，在体外和受试者中重组它们 一系列互补的结构检测方法(电子显微镜、共价交联 测绘和化学足迹)。这样发现的蛋白质-蛋白质相互作用将成为 基于结构的设计突变和多肽仿制。这些反过来将允许研究个人方面的 Rho在不影响其其他功能的情况下，作为基因表达的全球调节因子发挥作用。第二，我们要去 为了扩大我们对RNA介导的细胞内Rho功能调节的研究，增加新的靶点和 新的RNA类别(例如，非编码RNA)。通过部署下一代序列方法，我们将 为这些以前被忽视的监管机制生成一张全面的网络图。第三，在 除了Rho核蛋白质-蛋白质复合体的结构表征外，我们还将详细表征 Rho相互作用因子在体内和体内对其功能的影响，并阐明每种作用的机制。 我们的初步数据表明，这些生物(通常没有特征)可以使用多种机制。 调节剂，从简单的竞争性抑制Rho与RNA聚合酶的结合到其共价 修改(S)。 拟议研究的影响将是多方面的，就像它的方法一样。它将提供第一个 Rho作为细菌基因表达的全球调节者的全面和详细的图景。这一领域 转录调控将提供坚实的结构框架，以帮助解释现有数据 并指导未来的研究，以及将在其中部署的新工具。给定Rho表达的影响 水平转移的基因，包括致病岛，这项研究将对 了解细菌毒力的调节，而扰乱Rho功能的肽类药物可以发挥作用 作为抗菌药物开发中的新型先导化合物。

PROJECT SUMMARY/ABSTRACT ! This project aims at elucidating previously unknown aspects of termination factor Rho action in bacterial cell. We have discovered that Rho is a part of much larger than expected regulatory network that includes riboswitches and sRNAs. We also discovered a number of protein-protein interactions formed by Rho, independent of nucleic acids, allowing its effects on global gene expression to be modulated by the upstream regulatory signals. In this proposal we build upon these discoveries by deploying multi-faceted yet integrated analytical tools focusing on Rho interactions and non-canonical functions in the cell. First, we will use in vivo structural interactomics to discover protein complexes formed by Rho, reconstitute them in vitro and subject them to an array of complimentary methods of structural interrogation (electron microscopy, covalent cross-link mapping, and chemical footprinting). Protein-protein interactions thus discovered will then be targeted by structure-based designed mutations and peptidomimetics. These in turn will allow to study individual aspects of Rho action as a global regulator of gene expression without affecting its other functions. Second, we are going to expand our research of RNA-mediated regulation of Rho function in the cell by including new targets and new classes of RNA (e.g. non-coding RNAs). By deploying next generation sequences approaches we will generate a comprehensive network map of these previously overlooked regulatory mechanisms. Third, in addition to structural characterization of Rho-nucleated protein-protein complexes, we will characterize in detail the effects Rho-interactors have on its function in vivo and in vivo, and elucidate the mechanism of each effect. Our preliminary data indicates that a variety of mechanisms can be employed by these (often uncharacterized) regulators, from simple competitive inhibition of Rho association with RNA polymerase to its covalent modification(s). The impact of the proposed research will be as multi-faceted as its approach. It will provide the first comprehensive and detailed picture of Rho as a global regulator of gene expression in bacteria. The field of transcription regulation will be provided with solid structural framework to assist in interpreting existing data and directing future studies, as well as the novel tools to be deployed therein. Given Rho impact of expression of horizontally transferred genes, including pathogenicity islands, this research will have a positive impact on understanding of regulation of bacterial virulence, whereas peptidomimetics disrupting Rho function can serve as novel lead compounds in development of anti-bacterials.