Mechanism of CsrA-Mediated Global Control

描述（由申请人提供）：通过研究大肠杆菌的碳储存调节系统（Csr），将深入了解转录后调节机制和全球调节电路。Csr包括：CsrA，一种调节大量靶mRNA的翻译和/或稳定性的RNA结合蛋白; CsrB和Csrc，使用分子模拟来螯合和拮抗CsrA的非编码sRNA; BarA-UvrY，一种激活csrB和csrC转录的双组分信号转导系统;以及CsrD，一种特异性靶向CsrB和Csrc RNA以供RNA酶E降解的蛋白质。在大肠在大肠杆菌中，CsrA在广泛的范围内调节代谢、运动和多细胞行为。我们最近发表的RNA-seq研究确定了712种不同的RNA与CsrA结合，包括超过40种调节因子的mRNA。我们发表的和初步的研究结果进一步揭示，Csr与许多其他全球调节回路密切相关，这意味着Csr在全球范围内影响细菌生理学。我们推测，这种复杂的电路允许CsR在转录后水平上加强应激反应系统的转录效应。 本研究的具体目标是：1）通过基因筛选和RNA-seq研究，确定Csr与其他全局调控系统的相互调控作用，并阐明CsrA介导的新调控机制。我们将专注于CsRA介导的调节两个应激反应西格玛因子（<$S和<$E），和两个核糖核酸酶，参与批量mRNA周转（RNase E和PNTR）。2)定义CsrB/C RNA周转途径，重点是CsrD促进RNase E依赖性切割的机制。特别感兴趣的是，我们的初步结果表明，CsrD特异性耦合CsrB合成与靶向营业额的RNase E，这表明RNA合成对RNA衰变的影响可能是一个未被充分认识的RNA生物学特征。3)确定DeaD（一种DEAD盒RNA解旋酶）正调控CsrB/C RNA水平的分子机制。我们的初步结果表明，DeaD直接影响的反应调节UvrY，这是所需的csrB/C转录的表达。我们的研究结果进一步表明，DeaD解旋的二级结构，抑制uvrY的翻译。因此，这些研究将确定一个重要的监管影响Csr系统的机制，并提供基本的洞察DeaD解旋酶在翻译中的作用。 这些研究的长期目标是充分了解Csr系统的调控成分、遗传电路、分子机制和生物学功能，从而确定支撑调控超级网络的基本原则。与Csr同源的高度保守的调控系统控制许多人类、动物和植物病原体中的毒力因子和/或传播性状的表达，从而引起不同的感染。因此，这些研究将提供对细菌代谢和发病机制的调节的基本理解，并可能提出新的治疗和/或疫苗策略。

DESCRIPTION (provided by applicant): Insight into posttranscriptional regulatory mechanisms and global regulatory circuitry will be sought through the study of the carbon storage regulatory system (Csr) of Escherichia coli. Csr includes: CsrA, an RNA binding protein that regulates translation and/or the stability of a large number of target mRNAs; CsrB and CsrC, noncoding sRNAs that use molecular mimicry to sequester and antagonize CsrA; BarA-UvrY, a two- component signal transduction system that activates transcription of csrB and csrC; and CsrD, a protein that specifically targets CsrB and CsrC RNAs for degradation by RNase E. In E. coli, CsrA regulates metabolism, motility, and multicellular behavior on a broad scale. Our recently published RNA-seq studies identified 712 different RNAs that bind to CsrA, including mRNAs for >40 regulatory factors. Our published and preliminary findings further reveal that Csr is reciprocally linked to a number of other global regulatory circuits, implying tht Csr affects bacterial physiology on a global scale. We hypothesize that this complex circuitry allows Csr to reinforce the transcriptional effects of stress response systems at a posttranscriptional level. The specific aims of this proposal are: 1) Identify reciprocal regulatoy interactions of Csr with other global regulatory systems and elucidate novel CsrA-mediated regulatory mechanisms, which have come to light from the results of genetic screens and RNA-seq studies. We will focus on CsrA-mediated regulation of two stress-response sigma factors (¿S and ¿E), and two ribonucleases that participate in bulk mRNA turnover (RNase E and PNPase). 2) Define the CsrB/C RNA turnover pathway, focusing on the mechanism by which CsrD promotes RNase E-dependent cleavage. Of particular interest, our preliminary results indicate that CsrD specifically couples CsrB synthesis with targeted turnover by RNase E, suggesting that the influence of RNA synthesis on RNA decay may be an underappreciated feature of RNA biology. 3) Determine the molecular mechanisms by which DeaD, a DEAD-box RNA helicase, positively regulates CsrB/C RNA levels. Our preliminary results indicate that DeaD directly affects expression of the response regulator UvrY, which is required for csrB/C transcription. Our results further suggest that DeaD unwinds a secondary structure that inhibits translation of uvrY. Thus, these studies will determine the mechanism of an important regulatory influence on the Csr system and provide fundamental insight into the role of DeaD helicase in translation. The long-range objectives of these studies are to fully understand the regulatory components, genetic circuitry, molecular mechanisms, and biological functions of the Csr system, thereby defining basic principles that underpin a regulatory super-network. Highly conserved regulatory systems homologous to Csr control the expression of virulence factors and/or transmission traits in numerous human, animal, and plant pathogens, responsible for diverse infections. Thus, these studies will provide fundamental understanding of the regulation of bacterial metabolism and pathogenesis and may suggest novel therapeutic and/or vaccine strategies.