Molecular Determinants of Regulatory Hierarchy for Bacterial Small RNAs

项目总结 小RNA(SRNA)数量众多，执行复杂的调控机制，但它们在 细菌的生理和毒力大体上仍然是个谜。我们之前对两种模型大肠杆菌的研究 SRNAs，SGRs和RyhB，阐明了sRNAs在细菌生理和应激中所扮演的一些角色 反应和发现新的分子调控机制。我们证明了一个单一的sRNA可以 与许多mRNA靶标的碱基配对，并促进这些mRNAs的快速降解，具有重要的 对糖运输和代谢(SGRS)和铁稳态等关键过程的影响 (RyhB)。我们定义了一类新的竞争者，即所谓的“海绵”RNA，它结合和调节 调节其信使核糖核酸靶标的sRNA。在目前的建议中，我们在这些初步研究的基础上， 处理与SNA领域广泛相关的几个问题。1)定义SRNA的动力学参数是什么 与他们的目标互动？我们的初步数据表明，对于SGR，目标搜索(其中 Srna找到适当的目标)是速率限制，并且每个参数在数量上是不同的 不同的sRNA-靶相互作用。在目标1中，我们将使用一种新的超分辨率成像平台，我们 进一步研究SGR和RyHB与靶mRNAs的相互作用并定义一般原则 管理sRNA-信使核糖核酸相互作用。2)海绵RNA如何影响sRNA的调控效率 在很多不同的目标上？SRNAs的调控作用从对某些靶标的强烈影响到对某些目标的弱影响不等 在其他目标上。这建立了一个监管层级，我们认为这是srna介导的控制的核心 细菌应激反应。在目标2中，我们使用定量参数来定义sRNA调节效率和 确定这些参数受竞争的海绵RNA的存在和不存在的影响 具有与sRNA结合的mRNAs。3)海绵介导的调节对生理有什么影响 SRNA的活动以及这种控制水平有多普遍？我们的初步数据显示海绵 RNA有助于调节sRNA的活性，以便sRNA只能作用于适当的 压力条件。虽然发现的很少，但我们推测海绵RNA丰富，而且 它们可能被广泛用于控制许多细菌的sRNA。在目标3中，我们将解决这两个问题 通过研究海绵介导的控制SGRS和RyhB活性的生理后果以及 通过进行全基因组分析来识别和表征各种新的靶点和海绵RNA 细菌的sRNA。我们开发和优化的各种技术将使我们能够 在全球范围内和在单个RNA分子水平上询问sRNA与目标mRNAs的相互作用。 这将使我们能够生成由sRNA进行体内调控的定量模型，并阐明广泛的 SRNA调控网络产生比以往任何时候都更精确的SRNA相互作用图。

PROJECT SUMMARY Small RNAs (sRNAs) are numerous and carry out intricate regulatory mechanisms, yet their specific roles in bacterial physiology and virulence remain by and large mysterious. Our previous work with two model E. coli sRNAs, SgrS and RyhB, shed light on some of the roles played by sRNAs in bacterial physiology and stress responses and uncovered novel molecular mechanisms of regulation. We showed that a single sRNA can base pair with numerous mRNA targets, and promote the rapid degradation of these mRNAs, with important consequences for key processes including sugar transport and metabolism (SgrS) and iron homeostasis (RyhB). We defined a novel class of competitors, so-called “sponge” RNAs that bind and modulate the ability of sRNAs to regulate their mRNA targets. In the current proposal, we build on these preliminary studies to address several questions with broad relevance to the sRNA field. 1) What kinetic parameters define sRNA interactions with their targets? Our preliminary data indicate that for SgrS, the target search (where the sRNA finds the appropriate target) is rate limiting and that this parameter is quantitatively different for each distinct sRNA-target interaction. In Aim 1, we will use a novel super-resolution imaging platform that we developed to further interrogate SgrS and RyhB interactions with target mRNAs and define general principles governing sRNA-mRNA interactions. 2) How do sponge RNAs affect the regulatory efficiency of an sRNA on many different targets? Regulation by sRNAs varies from strong effects on some targets to weak effects on other targets. This sets up a regulatory hierarchy that we propose is central to sRNA-mediated control of bacterial stress responses. In Aim 2, we use quantitative parameters defining sRNA regulatory efficiency and determine how these parameters are impacted by the presence and absence of sponge RNAs, which compete with mRNAs for binding to sRNAs. 3) What are the physiological impacts of sponge-mediated regulation of sRNA activity and how prevalent is this level of control? Our preliminary data suggest that sponge RNAs help tune sRNA activity so that sRNAs are only able to act on target mRNAs under the appropriate stress conditions. Though very few have been discovered, we postulate that sponge RNAs are abundant, and that they may be widely used to control many bacterial sRNAs. In Aim 3, we will address both of these issues by examining the physiological consequences of sponge-mediated control of both SgrS and RyhB activity and by conducting genome-wide analyses to identify and characterize novel targets and sponge RNAs for a variety of bacterial sRNAs. The diverse set of techniques that we have developed and optimized will allow us to interrogate sRNA interactions with target mRNAs on a global scale and at the level of single RNA molecules. This will allow us to generate quantitative models for in vivo regulation by sRNAs and elucidate an extensive sRNA regulatory network to produce an sRNA interaction map of greater precision than ever before.