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

描述（由申请人提供）：小rna （sRNAs）已被推向遗传学研究的前沿，因为它们在细胞过程调节中的多种作用已被认识到。sRNAs对高等生物的发育至关重要；在细菌中，sRNAs调节对许多环境压力的反应。需要RNA伴侣Hfq的细菌sRNAs通过碱基配对依赖机制调节mRNA靶标的翻译或稳定性。hfq依赖的sRNA SgrS在大肠杆菌中对葡萄糖-磷酸盐胁迫的反应是必不可少的。SgrS代表了细菌中sRNA调节因子的新范式。其他特征的hfq依赖性sRNAs是非编码的，仅通过碱基配对和调节靶mrna（核糖调节）发挥作用。SgrS对mRNA靶点进行核糖调节，并编码一种新的蛋白质SgrT。我们的初步数据表明，SgrS的核糖调节功能比蛋白质编码功能更具有进化保守性。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，证明其中3个通过与SgrS的碱基配对直接上调或下调。对SgrS同源物及其推测靶点的分析表明，SgrS:mRNA相互作用在某些靶点上比其他靶点更保守。由于单个sRNA对多个靶点的调控是细菌和真核生物sRNA调控的共同主题，我们建议使用SgrS作为模型来研究具有不同碱基配对特征和亲和力的多个靶点的调控是如何协调的。Aim 1的实验将定义SgrS目标调控。在Aim 2中，高通量RNA足迹方法将用于绘制所有大肠杆菌SgrS:mRNA靶标和一些具有预测独特碱基配对特征的同源SgrS:mRNA对的sRNA:mRNA相互作用图谱。这些研究将证明SgrS与其靶点之间的直接相互作用，并确定这些相互作用的分子决定因素。在Aim 3中，我们将确定当SgrS水平有限时，SgrS是否存在优先调节目标的机制（即建立层次结构）。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。

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.