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

描述(由申请人提供)：小RNA(SRNA)已被推向遗传研究的前沿，因为它们在调节细胞过程中的不同作用已被认识到。SRNA对高等生物体的发育至关重要；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通过碱基配对依赖的机制调节mRNA靶标的翻译或稳定性。依赖于Hfq的SRNA SGRs在大肠杆菌对葡萄糖-磷酸胁迫的反应中是必不可少的。SGRS代表了细菌中sRNA调控的新范式。其他特征的依赖于Hfq的sRNAs是非编码的，仅通过碱基配对和调节靶mRNAs发挥作用(核调节)。SGRS对mRNA靶标进行核转录调节，并编码一种新的蛋白质SgrT。我们的初步数据表明，SGRS的核转录调节功能在进化上比蛋白质编码功能更保守。初步的微阵列实验确定了10个候选SGRS靶向mRNAs，我们提供了进一步的证据，其中3个直接通过与SGRS的碱基配对而上调或下调。对SGRS同源物及其可能的靶点的分析表明，SGRS：mRNA的相互作用在某些靶点上比其他靶点更保守。由于单个SRNA对多个靶点的调节是细菌和真核SRNA调节器的共同主题，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节是如何协调的。目标1中的实验将定义SGRS靶标调节子。在目标2中，将使用高通量RNA足迹方法来映射所有大肠杆菌SGRS：mRNA靶标和一些具有预测的独特碱基配对特征的同源SGRS：mRNA对的sRNA：mRNA相互作用。这些研究将证明SGRs与其靶标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。在目标3中，我们将确定是否存在一种机制，供SGR在SGRS水平有限时(即建立一个等级)优先对目标进行监管。然后，我们将通过突变改变碱基配对相互作用，并确定这些改变如何影响调控体系。 公共卫生相关性：由于从细菌到人类的sRNA被认为通过碱基配对依赖的机制调节多个靶点，这些研究具有广泛的相关性，因为它们将增强我们对sRNA介导的调控的基本特征的理解。对SGRs的研究尤其重要，因为SGRs是一类新的双功能sRNA调节器的第一个成员，因此可以作为未来将被确定的其他类似sRNA的模型。

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.