Analysis of the Molecular Determinants of Regulatory Hierarchy of a Bacterial Sma

细菌 Sma 调控层次的分子决定因素分析

• 批准号: 8115806
• 负责人: Carin K Vanderpool
• 金额: $ 29.26万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8115806
• 关键词: Address; Affect; Affinity; Bacteria; Base Composition; Base Pairing; Binding; Binding Sites; Cell physiology; Characteristics; Code; DNA Binding; Data; Development; Escherichia coli; Functional RNA; Future; Genetic; Genetic Research; Glucosephosphates; Homologous Gene; Human; Libraries; Light; Location; Maps; Mediating; Messenger RNA; Methods; Modeling; Molecular; Molecular Analysis; Molecular Chaperones; Monitor; Mutagenesis; Nature; Operator Regions; Organism; Physiology; Proteins; RNA; Regulation; Regulon; Research; Role; Signal Transduction; Small RNA; Stress; Structure; Translations; base; insight; mRNA Stability; member; novel; promoter; public health relevance; research study; response

DESCRIPTION (provided by applicant): Small RNAs (sRNAs) have been propelled to the forefront of genetic research as their diverse roles in regulation of cellular processes have been recognized. sRNAs are crucial for development in higher organisms; in bacteria, sRNAs regulate responses to many environmental stresses. Bacterial sRNAs that require the RNA chaperone Hfq regulate the translation or stability of mRNA targets by base pairing-dependent mechanisms. The Hfq-dependent sRNA SgrS is essential in E. coli for the response to glucose- phosphate stress. SgrS represents a new paradigm for sRNA regulators in bacteria. Other characterized Hfq-dependent sRNAs are non-coding and function solely via base pairing with and regulating target mRNAs (riboregulation). SgrS performs riboregulation on mRNA targets, and additionally encodes a novel protein, SgrT. This proposal focuses on the riboregulation function of SgrS, which our preliminary data indicates is more evolutionarily conserved than the protein coding function. Preliminary microarray experiments identified 10 candidate SgrS target mRNAs and we provide further evidence that 3 of these are directly up- or down-regulated by base pairing with SgrS. Analysis of SgrS homologs and their putative targets suggests that SgrS:mRNA interactions are better conserved for some targets than others. Since regulation of multiple targets by a single sRNA is a common theme for both bacterial and eukaryotic sRNA regulators, we propose to use SgrS as a model to examine how regulation of multiple targets with different base pairing characteristics and affinities is coordinated. Experiments in Aim 1, will define the SgrS target regulon. In Aim 2, a high- throughput RNA footprinting approach will be used to map sRNA:mRNA interactions for all E. coli SgrS:mRNA targets and some homologous SgrS:mRNA pairs with predicted unique base pairing characteristics. These studies will demonstrate direct interactions between SgrS and its targets and define the molecular determinants of these interactions. In Aim 3, we will determine whether there is a mechanism for SgrS to prioritize regulation of targets when SgrS levels are limiting (i.e., establish a hierarchy). We will then alter base pairing interactions by mutagenesis and determine how these alterations affect the regulatory hierarchy. PUBLIC HEALTH RELEVANCE: Since sRNAs from organisms from bacteria to humans are believed to regulate multiple targets by base pairing-dependent mechanisms, these studies are broadly relevant because they will enhance our understanding of basic characteristics of sRNA- mediated regulation. Studies of SgrS in particular are important since SgrS is the first member of a novel class of bifunctional sRNA regulators and therefore serves as a model for other similar sRNAs that will be identified in the future.

描述（由申请人提供）：小RNA（sRNA）已被推到遗传研究的最前沿，因为它们在细胞过程调节中的多种作用已被认识到。sRNA对高等生物的发育至关重要;在细菌中，sRNA调节对许多环境压力的反应。需要RNA伴侣Hfq的细菌sRNA通过碱基配对依赖性机制调节mRNA靶标的翻译或稳定性。 Hfq依赖的sRNA SgrS在大肠杆菌中是必需的。coli对葡萄糖-磷酸盐胁迫的反应。SgrS代表了细菌中sRNA调节剂的新范式。其他表征的Hfq依赖性sRNA是非编码的，并且仅通过与靶mRNA的碱基配对和调节靶mRNA（核糖核酸调节）发挥功能。SgrS对mRNA靶点进行核糖核酸调控，并编码一种新的蛋白质SgrT。这个建议的重点是核糖调节功能的SgrS，我们的初步数据表明，这是更进化保守的蛋白质编码功能。初步的微阵列实验确定了10个候选SgrS靶mRNA，我们提供了进一步的证据，其中3个直接上调或下调与SgrS的碱基配对。SgrS同源物和它们的推定目标的分析表明，SgrS：mRNA相互作用是更好地保守的一些目标比其他。由于由单个sRNA调节多个靶点是细菌和真核sRNA调节器的共同主题，因此我们建议使用SgrS作为模型来研究如何协调具有不同碱基配对特征和亲和力的多个靶点的调节。 目标1中的实验将定义SgrS靶调节子。在目标2中，高通量RNA足迹法将用于绘制所有大肠杆菌的sRNA：mRNA相互作用。coliSgrS：mRNA靶点和一些同源的SgrS：mRNA对，具有预测的独特碱基配对特征。这些研究将证明SgrS与其靶标之间的直接相互作用，并定义这些相互作用的分子决定因素。在目标3中，我们将确定当SgrS水平受限时（即，建立一个层次）。然后，我们将通过诱变改变碱基配对相互作用，并确定这些改变如何影响调控层次。 公共卫生相关性：由于来自从细菌到人类的生物体的sRNA被认为通过碱基配对依赖性机制调节多个靶标，因此这些研究具有广泛的相关性，因为它们将增强我们对sRNA介导的调节的基本特征的理解。SgrS的研究特别重要，因为SgrS是一类新的双功能sRNA调节子的第一个成员，因此可以作为未来将鉴定的其他类似sRNA的模型。