Quantitative Imaging and Modeling of Regulation by Bacterial Small RNA

描述（由申请人提供）：细菌小rna（这里称为sRNAs）是基因表达的重要调控因子，特别是那些与应激反应和毒力相关的基因。sRNAs通过与目标mrna的碱基配对来发挥功能，并影响其翻译和稳定性。每个sRNA在其调控中调节多个靶标的能力创建了一个复杂的网络，用于塑造基因表达和表型，最终导致细菌的全球适应。srna介导调控的动力学特性如何建立有序的基因表达模式？srna介导的调控对细菌表型的全球影响是什么？这是有待解决的两个重要的基本问题。在本提案中，我们将解决这些问题。迄今为止，阻碍建立一个完整的模型来描述sRNA调控网络的瓶颈之一是缺乏细菌细胞内部的动力学测量。因此，我们首先提出开发一种超分辨率成像和分析平台，允许在单细胞水平上以单拷贝灵敏度直接可视化和表征sRNA和靶mrna。将该成像分析平台应用于大肠杆菌模型sRNA系统SgrS，我们将全面剖析sRNA对ai1中单个靶点调控的动力学机制，并进一步探索ai2中调控中多个靶点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Aim 3中，我们将整合高通量转录组学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，并确定sRNA调控导致的代谢途径使用差异。该研究结合了多层次的实验表征和计算模拟，将提供迄今为止最系统的sRNA调控描述，并将建立一个新的sRNA分析框架，可推广到其他细菌和真核生物的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.