Mechanism of CsrA-Mediated Global Control

描述(由申请人提供)：通过对大肠杆菌碳储存调控系统(CSR)的研究，将寻求对转录后调控机制和全球调控电路的洞察。CSR包括：CSRA，一种RNA结合蛋白，调节大量靶mRNAs的翻译和/或稳定性；CsrB和CSRC，非编码sRNA，使用分子模仿来隔离和拮抗CSRA；Bara-UvrY，一个双组分信号转导系统，激活csrB和CSRC的转录；以及CsrD，一种专门针对CsrB和CSRC RNA的蛋白质，可被RNase E降解。在大肠杆菌中，CSRA调节新陈代谢、运动性和广泛的多细胞行为。我们最近发表的RNA-SEQ研究确定了712个不同的RNA与CSRA结合，包括40个调控因子的mRNAs。我们已发表的和初步的研究结果进一步表明，CSR与其他一些全球调控电路相互关联，这意味着CSR在全球范围内影响细菌生理。我们假设，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。这一建议的具体目的是：1)确定CSR与其他全球调控系统的相互调控相互作用，并阐明CSRA介导的新的调控机制，这些机制已经从遗传筛选和RNA-SEQ研究的结果中揭示出来。我们将集中于CsrA介导的两个应激反应Sigma因子(�S和�E)和两个参与大宗mR-NA转换的核糖核酸酶(RNaseE和PNPase)的调节。2)定义CsrB/C RNA转换途径，重点研究CsrD促进RNase E依赖的切割的机制。特别有趣的是，我们的初步结果表明，CsrD特异性地将CsrB的合成与RNase E的靶向翻转结合在一起，这表明RNA合成对RNA衰退的影响可能是RNA生物学中一个被低估的特征。3)确定DEAD(DEAD-box RNA解旋酶)正向调节CsrB/C RNA水平的分子机制。我们的初步结果表明，DEAD直接影响反应调节因子UvrY的表达，而UvrY是csrB/C转录所必需的。我们的结果进一步表明，DEAD解开了抑制uvrY翻译的二级结构。因此，这些研究将确定对CSR系统产生重要调控影响的机制，并为深入了解死亡解旋酶在翻译中的作用提供基础。这些研究的长期目标是全面了解CSR系统的调节成分、遗传电路、分子机制和生物功能，从而确定支撑调节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着许多人类、动物和植物病原体中毒力因子和/或传播特性的表达，这些病原体导致了不同的感染。因此，这些研究将提供对细菌代谢调节和发病机制的基本了解，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.