Characterization of a novel role for DEAD-box RNA helicases in bacteria.

描述(由申请者提供)：本项目的长期目标是加强对基因调控转录后机制的基本理解。本文研究的碳储存调控系统CSR对β-蛋白细菌的生物膜的形成、运动和毒力因子的表达有着深远的影响。拟议的研究详细说明了一项计划，以调查死盒RNA解旋酶在CSR调控中的作用。DEAD-box RNA解旋酶是一个知之甚少的调节蛋白家族，其在细菌中的作用被认为仅限于冷应激期间rRNA的成熟，并在RNA周转中辅助次级切割事件。相反，这项研究解决了死盒RNA解旋酶的调节作用，a)影响控制许多细菌毒力因子的全球调节系统的表达，以及b)发生在与哺乳动物感染相关的温度(37℃)。CSRA是一种RNA结合蛋白，通过与mRNAs的5‘非翻译区(5’UTR)结合，在转录后调节许多重要的细菌基因和系统，从而改变蛋白质翻译和/或RNA周转。非编码RNA CsrB和CSRC以高亲和力结合多个CSRA蛋白，从而隔离和拮抗该蛋白。这些ncRNAs的转录依赖于BarA/UvrY双组分信号转导系统。现有数据表明，BARA/UvrY回路是将环境刺激传递给CSR调节的中央输入机制。转座子突变筛选发现，大肠杆菌DEAD-box RNA解旋酶SrmB和DEAD是CsrB表达的强烈调控因子。SrmB和Dead似乎通过BarA/UvrY系统独立地影响CsrB的表达。死亡缺失突变体显著降低了UvrY水平，而srmB缺失突变体表现出BarA/UvrY磷酸化降低，而不影响这些蛋白的水平。这项提议的重点是阐明Dead和SrmB调控Bara/UvrY的不同途径。这项建议的具体目的是确定(I)死亡和(Ii)srB是如何独立调节CsrB水平并最终调节CSRA活性的。我们将检验DEAD通过促进UvrY翻译而增加UvrY蛋白水平的假设。我们将结合体内磷酸化实验和上位性实验来进一步表征SrmB对UvrY磷酸化的影响。最后，我们将利用遗传筛选来确定这一途径中的因素。 这项建议的目标有三个：i)确定死盒RNA解旋酶影响CSR活性的机制；ii)确定一类RNA解旋酶的新的体内靶点，其主要活性被认为是在冷休克下加工rRNA；以及iii)建立一个模型系统来研究在原核生物、真核生物和病毒中具有不同功能的一类重要的生物分子。

DESCRIPTION (provided by applicant): The long-term objective of this project is to enhance the fundamental understanding of post- transcriptional mechanisms of gene regulation. The carbon storage regulatory system, "Csr", studied herein, has profound effects on biofilm formation, motility, and the expression of virulence factors in ?-proteobacteria. The proposed studies detail a plan to investigate the role of DEAD-box RNA helicases in Csr regulation. The DEAD-box RNA helicases constitute a poorly understood family of regulatory proteins, whose role in bacteria was thought to be limited to rRNA maturation during cold stress, and assisting secondary cleavage events in RNA turnover. In contrast, this study addresses regulatory roles for DEAD-box RNA helicases that a) influence the expression of a global regulatory system controlling a number of bacterial virulence factors, and b) occur at temperatures relevant for mammalian infections (37¿C). CsrA is an RNA-binding protein that post-transcriptionally regulates numerous important bacterial genes and systems by binding to the 5' untranslated region (5' UTR) of mRNAs, thus altering protein translation and/or RNA turnover. The non-coding RNAs CsrB and CsrC bind multiple CsrA proteins with high affinity, and thereby sequester and antagonize this protein. Transcription of these ncRNAs depends upon the BarA/UvrY two-component signal transduction system. Available data suggest that the BarA/UvrY circuitry is the central input mechanism for transmitting environmental stimuli to regulation of Csr. A transposon mutagenesis screen identified the E. coli DEAD-box RNA helicases SrmB and DeaD as strong regulators of CsrB expression. SrmB and DeaD appear to affect CsrB expression independently through the BarA/UvrY system. A DeaD deletion mutant has significantly reduced UvrY levels whereas an srmB deletion exhibits reduced phosphorylation of BarA/UvrY without affecting the levels of these proteins. The focus of this proposal is to elucidate the separate pathways by which DeaD and SrmB regulate BarA/UvrY. The specific aims of this proposal are to determine how (i) DeaD and (ii) SrmB independently regulate CsrB levels and ultimately modulate CsrA activity. We will test the hypothesis that DeaD increases UvrY protein levels by promoting uvrY translation. We will combine in vivo phosphorylation assays with epistasis experiments to further characterize the effect of SrmB on UvrY phosphorylation. Finally, we will utilize genetic screens to identify factors in this pathway. The goals of this proposal are three-fold: i) determine the mechanisms by which DEAD-box RNA helicases affect Csr activity, ii) define novel in vivo targets of a class of RNA helicases whose primary activity was thought to be rRNA processing under cold- shock, and iii) develop a model system for the study of an important class of biomolecules that have diverse functions in prokaryotes, eukaryotes, and viruses.