Systematic analysis of small RNA-based regulation of gene expression in bacteria

基于小RNA的细菌基因表达调控的系统分析

• 批准号: 9212943
• 负责人: Nicholas R. De Lay
• 金额: $ 32.34万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-03 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9212943
• 关键词: 5' -exoribonuclease; Active Sites; Antibiotic Resistance; Bacteria; Base Pairing; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; C-terminal; Cleaved cell; Co-Immunoprecipitations; Data; Development; Endoribonucleases; Enzymes; Escherichia coli; Eukaryota; Exoribonuclease II; Exoribonucleases; Family; Gene Expression; Gene Expression Regulation; Genetic; Goals; Growth; Human; In Vitro; Knowledge; Lead; Maps; Measures; Mediating; Messenger RNA; Metabolism; Methodology; Microbial Biofilms; Molecular; Molecular Chaperones; Mutation Analysis; Organism; Play; Polyribonucleotide Nucleotidyltransferase; Post-Transcriptional Regulation; Printing; Process; Public Health; RNA; RNA Binding; RNA Decay; RNA Degradation; RNA Helicase; RNA Sequences; RNA Stability; Recruitment Activity; Research; Ribonucleases; Role; Site; Small RNA; Structure; Substrate Specificity; System; Testing; Translations; Untranslated RNA; Virulence; base; biological adaptation to stress; cell motility; enolase; experimental study; foot; in vivo; innovation; insight; mRNA Decay; mRNA Stability; novel; pathogen; resistance mechanism; ribonuclease E; ribonuclease R; stem

Project Summary/Abstract There are fundamental gaps in our knowledge about how bacterial small, noncoding RNAs (sRNAs) find and base-pair with a target mRNA and how this pairing leads to changes in gene expression. The long- term goal of this research is to elucidate the mechanisms of post-transcriptional regulation of gene expression in bacteria. The overall objective of this proposal is to understand how PNPase controls RNA stability and decay. Our central hypothesis is that PNPase plays a key role in gene regulation by protecting Hfq-bound sRNAs from degradation, degrading unbound sRNAs, and targeting paired sRNAs and mRNAs to the RNA degradosome for degradation. The experiments described herein will test this hypothesis and further define the molecular mechanisms by which PNPase binds, protects, and degrades RNAs using E. coli PNPase as the model system. The significance of the proposed research is that it will advance our knowledge of a novel activity of PNPase, stabilizing RNAs, and increase our understanding of sRNA-mediated regulation of gene expression, which is integral to bacterial stress responses and include antibiotic resistance mechanisms. The research proposed in this application is innovative, because it will challenge the existing paradigm that describes PNPase solely as an RNA degrading enzyme that only recognizes the 3' ends of RNAs. In Aim 1, we will define the molecular mechanism of PNPase-mediated RNA protection. Our working hypothesis is that PNPase protects Hfq-bound sRNAs from degradation by other RNases by occluding potential binding or cleavage sites. Using genetic, molecular, and biochemical approaches we will define the sites of interactions between PNPase and sRNAs, test the role of the exoribonuclease activity of PNPase in sRNA protection, and assess the contribution of other RNases to sRNA decay in the absence of PNPase. In Aim 2, the mechanism by which PNPase mediates decay of sRNAs and target mRNAs will be investigated. Our working hypothesis is that PNPase degrades sRNAs not bound to Hfq, and targets certain paired sRNAs and mRNAs to the RNA degradosome for degradation. The RNA degradosome is comprised of the endoribonuclease RNase E, glycolytic enzyme enolase, the RNA helicase RhlB, and PNPase. Through a comprehensive set of genetic, molecular, and biochemical approaches, we will define the substrate specificity of PNPase and the role of particular residues in the degradation of RNAs. Finally, we will define the sites of RNase E that interact with sRNAs and test whether or not PNPase contributes to recruitment of RNAs to RNase E upon sRNA-mRNA pairing. Since PNPase is highly conserved among bacteria and eukaryotes, understanding the molecular mechanism of how PNPase controls RNA stability in E. coli will provide insight into RNA metabolism in most living organisms.

项目概要/摘要 我们对于细菌小非编码 RNA (sRNA) 如何发挥作用的认识存在根本性差距 找到目标 mRNA 并进行碱基配对，以及这种配对如何导致基因表达的变化。长- 这项研究的长期目标是阐明基因表达转录后调控的机制 在细菌中。该提案的总体目标是了解 PNPase 如何控制 RNA 稳定性和 衰变。我们的中心假设是 PNPase 通过保护 Hfq 结合在基因调控中发挥关键作用 防止 sRNA 降解、降解未结合的 sRNA，以及将配对的 sRNA 和 mRNA 靶向 RNA 降解体用于降解。本文描述的实验将检验这一假设并进一步定义 使用大肠杆菌 PNPase 作为 PNPase 结合、保护和降解 RNA 的分子机制 模型系统。这项研究的意义在于它将增进我们对小说的了解 PNPase 的活性，稳定 RNA，并增加我们对 sRNA 介导的基因调控的理解 表达，这是细菌应激反应不可或缺的一部分，包括抗生素耐药机制。这 本申请中提出的研究具有创新性，因为它将挑战现有的范式 PNPase 仅描述为一种 RNA 降解酶，仅识别 RNA 的 3' 末端。在目标 1 中， 我们将定义 PNPase 介导的 RNA 保护的分子机制。我们的工作假设是 PNPase 通过阻断潜在的结合或保护 Hfq 结合的 sRNA 免受其他 RNase 的降解 切割位点。使用遗传、分子和生化方法，我们将定义相互作用的位点 PNPase 和 sRNA 之间的关系，测试 PNPase 的核糖核酸外切酶活性在 sRNA 保护中的作用，以及 评估在没有 PNPase 的情况下其他 RNase 对 sRNA 衰减的贡献。在目标 2 中，该机制 将研究 PNPase 介导 sRNA 和靶 mRNA 的衰变。我们的工作假设是 PNPase 降解未与 Hfq 结合的 sRNA，并将某些配对的 sRNA 和 mRNA 靶向该 RNA 降解体用于降解。 RNA 降解体由核糖核酸内切酶 RNase E 组成， 糖酵解酶烯醇酶、RNA 解旋酶 RhlB 和 PNPase。通过一套全面的遗传基因， 通过分子和生化方法，我们将定义 PNPase 的底物特异性以及 RNA 降解过程中的特殊残基。最后，我们将定义与 RNase E 相互作用的位点 sRNA 并测试 PNPase 是否有助于在 sRNA-mRNA 上将 RNA 募集至 RNase E 配对。由于 PNPase 在细菌和真核生物中高度保守，因此了解分子 PNPase 如何控制大肠杆菌中 RNA 稳定性的机制将有助于深入了解大多数细胞中的 RNA 代谢 活的有机体。