Molecular Determinants of Regulatory Hierarchy for Bacterial Small RNAs

项目摘要 小RNA（sRNA）数量众多，并执行复杂的调控机制，但它们在细胞内的特定作用， 细菌的生理学和毒性大体上仍然是个谜。我们以前的工作与两个模型E。杆菌 sRNAs，SgrS和RyhB，揭示了sRNAs在细菌生理学和应激中所起的一些作用 反应和发现新的分子调控机制。我们发现单个sRNA可以 与许多mRNA靶点配对，并促进这些mRNA的快速降解， 对糖转运和代谢（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，它们结合并调节蛋白质的表达能力。 sRNAs调节其mRNA靶点。在目前的建议中，我们在这些初步研究的基础上， 解决与sRNA领域广泛相关的几个问题。1)什么动力学参数定义sRNA 与目标的互动？我们的初步数据表明，对于SgrS，目标搜索（其中 sRNA找到合适的靶标）是速率限制性的，并且该参数对于每一种sRNA来说在数量上是不同的。 不同的sRNA-target相互作用。在Aim 1中，我们将使用一种新型的超分辨率成像平台， 开发用于进一步询问SgrS和RyhB与靶mRNA的相互作用并定义一般原则 控制sRNA-mRNA相互作用。2)海绵RNA如何影响sRNA的调节效率 针对不同的目标sRNA的调节作用从对某些靶点的强作用到弱作用不等 其他目标。这建立了一个调控层次，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，我们使用定义sRNA调控效率的定量参数， 确定这些参数如何受到海绵RNA的存在和不存在的影响， 用mRNA与sRNA结合。3)海绵介导的调节有哪些生理影响 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活性，使得sRNA仅能够在适当的条件下作用于靶mRNA。 应力条件虽然很少被发现，我们假设海绵RNA是丰富的， 它们可能被广泛用于控制许多细菌sRNA。在目标3中，我们将解决这两个问题 通过检查海绵介导的SgrS和RyhB活性控制的生理后果， 通过进行全基因组分析，以确定和表征各种新的靶标和海绵RNA， 细菌sRNAs的。我们开发和优化的各种技术将使我们能够 在全球范围内和单个RNA分子水平上询问sRNA与靶mRNA的相互作用。 这将使我们能够生成sRNA体内调控的定量模型，并阐明广泛的 sRNA调控网络，以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.