Systems approach to study sRNAs in Escherichia coli

我的NSERC研究项目的总体目标是了解细菌中小的非编码RNA(SRNA)对细胞应激的作用。我建议将高通量和分子生物学方法结合起来，研究sRNA介导的基因调控如何有助于模式细菌大肠杆菌的生长和应激反应。大肠杆菌表达约200个sRNA，其中大部分编码在基因组的间隔区或嵌入蛋白质编码基因的未翻译部分。SRNA基因通常是由生长环境的变化引起的，例如，营养胁迫或化学扰动。一旦表达，sRNAs通过与部分互补的mRNAs碱基配对，改变翻译和/或RNA的稳定性来调节蛋白质的合成。sRNAs的基因调控被认为是细菌应激反应的关键。矛盾的是，破坏sRNA介导的基因调控导致细菌生长或存活的明显表型的例子寥寥无几。事实上，我们对sRNA功能的大部分知识都来自于对少数sRNA的深入研究，留下了大多数人不知道的功能。近年来，我的团队率先开发了用于大规模研究大肠杆菌基因功能的工具。我们开发了一套仪器和软件，用于测量高密度菌落阵列中的细菌生长速度。我们还创造了一种成像设备，由3D打印外壳、单板计算机、相机和软件组成，用于研究菌落阵列中的荧光。这些工具使我们在了解基因功能的系统方法中处于独特的地位，我们通常使用化学或遗传扰动。到目前为止，我们的努力仅集中在有助于细菌健康的蛋白质编码基因上。在这项拟议的研究中，我们将致力于了解sRNA对细菌生长和细胞应激反应的重要性。目前细菌基因组的暗物质，我们的目标是描述一个全基因组范围的功能网络的sRNA在大肠杆菌。短期目标包括：*1.探索细菌细胞应激中sRNA的功能网络*2.检验sRNA功能网络提出的假说*3.测量不同生长条件下sRNA表达的动态*虽然迄今旨在了解sRNA功能的方法大多是简约主义的，但所提出的方法将使我们能够询问大量的扰动及其组合。这些将包括通过一次删除一个或多个sRNA基因而导致的基因突变，以及不同的环境压力。此外，我们还将以高时间分辨率分析数百种条件下sRNA启动子的转录情况。这一系统方法将为使用传统的生化和分子遗传学方法进行详细研究提供新的假设。总而言之，我们将解决在理解细菌sRNA对细胞应激的重要性方面的一个主要知识差距。

The overall objective of my NSERC research program is to understand the role of small non-coding RNAs (sRNA) to cell stress in bacteria. I propose to combine high-throughput and molecular biology approaches to study how sRNA-mediated gene regulation contributes to the growth and stress responses of the model bacterium, Escherichia coli.******E. coli expresses some 200 sRNAs, most of which are encoded in intergenic regions of the genome or embedded within untranslated portions of protein-coding genes. sRNA genes are typically induced by changes in the growth environment due, for example, to nutrient stress or chemical perturbants. Once expressed, sRNAs regulate protein synthesis by base-pairing to partially complementary mRNAs and altering translation and/or RNA stability.******Gene-regulation by sRNAs is thought to be vital to bacterial stress responses. Paradoxically, there are few examples where disruption of sRNA-mediated gene regulation results in a discernable phenotype in bacterial growth or survival. Indeed, much of our knowledge of sRNA function comes from in-depth studies on a small handful of sRNAs, leaving the function of most unknown.******In recent years, my group has pioneered the development of tools for large-scale investigations of gene function in E. coli. We developed a suite of instrumentation and software for measuring bacterial growth rates in high-density colony arrays. We have also created an imaging device, comprised of a 3D-printed housing, single board computer, camera and software, for studying fluorescence in colony arrays. These tools have us uniquely positioned for success in systems approaches to understanding gene function where we typically use chemical or genetic perturbation. To date, our efforts have focused exclusively on protein-coding genes that contribute to bacterial fitness. In the proposed research we will work to understand the importance of sRNAs to bacterial growth and cell stress responses. Currently the dark matter of bacterial genomes, we aim to describe a genome-wide functional network for sRNAs in E. coli. Short-term objectives include:******1. Probe the functional network of sRNAs in bacterial cell stress***2. Test hypotheses posed by the sRNA functional network***3. Measure sRNA expression dynamics in different growth conditions******While approaches to date aimed at understanding sRNA function have largely been reductionist, the proposed approach will allow us to interrogate a large number of perturbations and combinations thereof. These will include genetic mutations, through deletion of one or more sRNA genes at a time, and diverse environmental stresses. In addition we will profile transcription of sRNA promoters across hundreds of conditions with high temporal resolution. This systems approach will provide new hypotheses for detailed study using conventional biochemical and molecular genetic methods. In all we will address a major knowledge gap in understanding the importance of bacterial sRNAs to cell stress.**