Quantitative Imaging and Modeling of Regulation by Bacterial Small RNA

描述(申请人提供)：细菌小RNA(这里称为sRNAs)是基因表达的重要调节者，特别是那些与应激反应和毒力相关的基因表达。SRNAs通过碱基配对与靶mRNAs结合，影响其翻译和稳定性。每个sRNA调控其调节子中的多个靶点的能力创建了一个复杂的网络来塑造基因表达和表型，这最终导致细菌的全球适应。SRNA介导的调控的动力学特性如何建立基因表达的有序模式？SRNA介导的调控对细菌表型的全球影响是什么？这是两个尚未解决的重要根本性问题。在这项提案中，我们将解决这些问题。到目前为止，阻碍建立描述sRNA调控网络的完整模型的瓶颈之一是缺乏细菌细胞内的动力学测量。因此，我们首先提出开发一个超分辨率成像和分析平台，可以在单细胞水平上以单拷贝敏感性直接可视化和表征sRNA和靶mRNAs。将这个成像和分析平台应用于大肠杆菌中的一个模型sRNA系统SGRS，我们将全面剖析SRNA对Aim1中单个靶点的调控动力学机制，并在目标2中进一步探索调控子中多个靶点之间调控选择性的分子机制。为了了解SRNA调控对细菌表型的整体影响，在目标3中，我们将高通量转录数据和单细胞成像数据与大肠杆菌代谢的基因组尺度通量平衡模型相结合，识别由于SRNA调控而导致的不同代谢途径的使用。这项通过多水平实验表征和计算模拟相结合的研究将提供迄今为止对sRNA调控最系统的描述，并将建立一个新的sRNA分析框架，可推广到其他细菌和真核sRNA。

DESCRIPTION (provided by applicant): Bacterial small RNAs (here referred to as sRNAs) are important regulators for gene expression, especially those associated with stress responses and virulence. sRNAs function by base pairing with their target mRNAs and affecting their translation and stability. The ability of each sRNA to regulate multiple targets in its regulon creates a complex network for shaping gene expression and phenotype, which ultimately leads to global adaptation of bacteria. How do the kinetic properties of sRNA-mediated regulation establish an ordered pattern of gene expression? What is the global impact of sRNA-mediated regulation on bacterial phenotypes? These are two important fundamental questions yet to be addressed. In this proposal, we will tackle these questions. One of the bottlenecks so far that has precluded the building a complete model to describe sRNA regulatory networks is the lack of kinetic measurements inside bacterial cells. Therefore, we first propose to develop a super-resolution imaging and analysis platform allowing direct visualization and characterization of sRNA and target mRNAs at the single-cell level with single copy sensitivity. Applying this imaging and analysis platform to a model sRNA system, SgrS, in E. coli, we will fully dissect kinetic mechanisms of sRNA regulation on individual targets in Aim1, and further explore the molecular mechanism that governs the regulation selectivity among multiple targets in the regulon in Aim 2. In order to understand the global impact of sRNA regulation on the bacterial phenotype, in Aim 3, we will integrate high-throughput transcriptomic data and single cell imaging data with genome scale flux balance models of E. coli metabolism and identify differential metabolic pathway usage as a result of sRNA regulation. The proposed study by the combination of multi-level experimental characterization and computational simulation will provide the most systematic description of sRNA regulation to date and will establish a novel framework for sRNA analysis that can be generalized to other bacterial and eukaryotic sRNAs.