Transcription termination and gene regulation by Rho: Integrative analysis

Rho 的转录终止和基因调控：整合分析

• 批准号: 10152645
• 负责人: EVGENY A NUDLER
• 金额: $ 56.43万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10152645
• 关键词: 5' Untranslated Regions; Address; Affect; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacterial Genes; Biological Assay; Cells; Chemicals; Code; Complex; Cryoelectron Microscopy; DNA-Directed RNA Polymerase; Data; Development; Disease; Electron Microscopy; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome Stability; Horizontal Gene Transfer; Immune; In Vitro; Individual; Infection; Maps; Mass Spectrum Analysis; Mediating; Messenger RNA; Methods; Modeling; Modification; Molecular; Motor Activity; Mutation; Nucleic Acids; Pathogenicity; Pathogenicity Island; Physiological; Play; Proteins; RNA; RNA Helicase; Regimen; Regulation; Regulator Genes; Reporter; Research; Research Proposals; Resolution; Rho Factor; Role; Signal Transduction; Solid; Stress; Structure; Transcriptional Regulation; Untranslated RNA; Virulence; Work; analytical tool; antimicrobial; base; crosslink; design; drug development; genome-wide; human pathogen; in vivo; next generation; next generation sequencing; novel; novel lead compound; oxidation; peptidomimetics; protein complex; protein protein interaction; reconstitution; rho; single molecule; small molecular inhibitor; small molecule inhibitor; termination factor; tool; transcription termination; transcriptome

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.

项目概要/摘要 ！ 该项目旨在阐明细菌细胞中终止因子 Rho 作用的先前未知方面。 我们发现 Rho 是比预期大得多的监管网络的一部分，其中包括 核糖开关和 sRNA。我们还发现了 Rho 形成的许多蛋白质-蛋白质相互作用， 独立于核酸，允许其对全局基因表达的影响由上游调节 监管信号。在本提案中，我们通过部署多方面且集成的技术来建立在这些发现的基础上 专注于细胞中 Rho 相互作用和非规范功能的分析工具。首先，我们将使用vivo 结构相互作用组学，发现 Rho 形成的蛋白质复合物，在体外和受试者中重建它们 他们对结构询问的一系列补充方法（电子显微镜，共价交联 绘图和化学足迹）。由此发现的蛋白质-蛋白质相互作用将被靶向 基于结构的设计突变和肽模拟物。这些反过来又将允许研究的各个方面 Rho 作为基因表达的全局调节因子，而不影响其其他功能。第二，我们要去 通过纳入新靶点和 新类别的 RNA（例如非编码 RNA）。通过部署下一代序列方法，我们将 生成这些以前被忽视的监管机制的全面网络图。第三，在 除了 Rho 有核蛋白质-蛋白质复合物的结构表征外，我们还将详细表征 研究Rho相互作用物对其体内和体内功能的影响，并阐明每种作用的机制。 我们的初步数据表明，这些可以采用多种机制（通常没有特征） 调节剂，从 Rho 与 RNA 聚合酶结合的简单竞争性抑制到其共价 修改。 拟议研究的影响将与其方法一样是多方面的。它将提供第一个 全面详细地了解 Rho 作为细菌基因表达的全局调节因子。领域 将为转录调控提供坚实的结构框架，以协助解释现有数据 并指导未来的研究，以及其中部署的新颖工具。考虑到 Rho 表达的影响 水平转移基因，包括致病岛，这项研究将对 了解细菌毒力的调节，而破坏 Rho 功能的肽模拟物可以发挥作用 作为开发抗菌药物的新型先导化合物。

项目成果

Crucial role and mechanism of transcription-coupled DNA repair in bacteria.

• DOI: 10.1038/s41586-022-04530-6
• 发表时间: 2022-04
• 期刊: Nature
• 影响因子: 64.8

Pervasive Transcription-coupled DNA repair in E. coli.

• DOI: 10.1038/s41467-022-28871-y
• 发表时间: 2022-03-30
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Martinez B;Bharati BK;Epshtein V;Nudler E
• 通讯作者: Nudler E

Towards the unified principles of transcription termination.

走向转录终止的统一原则。

• DOI: 10.15252/embj.2019104112
• 发表时间: 2020
• 期刊: The EMBO journal
• 作者: Svetlov,Vladimir;Nudler,Evgeny
• 通讯作者: Nudler,Evgeny

High-resolution landscape of an antibiotic binding site.

• DOI: 10.1038/s41586-023-06495-6
• 发表时间: 2023-10
• 期刊: NATURE
• 影响因子: 64.8
• 作者: Yang, Kevin B.;Cameranesi, Maria;Gowder, Manjunath;Martinez, Criseyda;Shamovsky, Yosef;Epshtein, Vitaliy;Hao, Zhitai;Nguyen, Thao;Nirenstein, Eric;Shamovsky, Ilya;Rasouly, Aviram;Nudler, Evgeny
• 通讯作者: Nudler, Evgeny

RNA polymerase drives ribonucleotide excision DNA repair in E. coli.

• DOI: 10.1016/j.cell.2023.04.029
• 发表时间: 2023-05-25
• 期刊: CELL
• 影响因子: 64.5
• 作者: Hao, Zhitai;Gowder, Manjunath;Proshkin, Sergey;Bharati, Binod K.;Epshtein, Vitaly;Svetlov, Vladimir;Shamovsky, Ilya;Nudler, Evgeny
• 通讯作者: Nudler, Evgeny