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

项目摘要/摘要 我们对细菌小的非编码RNA(SRNA)的了解存在着根本性的空白 寻找并与目标mRNA进行碱基配对，以及这种配对如何导致基因表达的变化。长的- 本研究的长期目标是阐明基因表达的转录后调控机制。 在细菌中。这项提案的总体目标是了解PNPase如何控制RNA的稳定性和 腐烂。我们的中心假设是PNPase通过保护Hfq结合在基因调控中起关键作用 来自降解的sRNA，降解未结合的sRNA，并将成对的sRNA和mRNA靶向RNA 用于降解的降解体。这里描述的实验将检验这一假设，并进一步定义 PNPase结合、保护和降解RNA的分子机制 模型系统。拟议中的研究的意义在于它将促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，这是细菌应激反应的组成部分，并包括抗生素耐药机制。这个 本申请中提出的研究具有创新性，因为它将挑战现有的 将PNPase描述为仅识别RNA的3‘端的RNA降解酶。在目标1中， 我们将明确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RNA酶的降解 裂解位点。使用遗传、分子和生化方法，我们将定义相互作用的位置 在PNPase和sRNAs之间，测试PNPase的外切核酸酶活性在sRNA保护中的作用，以及 在没有PNPase的情况下，评估其他核糖核酸酶对sRNA衰退的贡献。在目标2中，机制 PNPase介导sRNAs和靶mRNAs衰变的机制将被研究。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RNA 用于降解的降解体。RNA降解体由内切核酸酶RNaseE组成， 糖酵解酶烯醇化酶、RNA解旋酶Rh1B和PNPase。通过一套全面的基因， 分子和生化方法，我们将定义底物专一性的PNPase和作用 RNA降解过程中的特殊残留物。最后，我们将定义与RNaseE相互作用的位置 并测试PNPase是否有助于RNase E在sRNA-mRNA上的RNAs招募 配对。由于PNPase在细菌和真核生物中高度保守，因此了解 PNPase在大肠杆菌中控制RNA稳定性的机制将为深入了解大多数 活着的有机体。

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.