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

DEAD-box RNA 解旋酶在细菌中的新作用的表征。

• 批准号: 8588782
• 负责人: Christopher Anthony Vakulskas
• 金额: $ 5.51万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8588782
• 关键词: 5' Untranslated Regions; Address; Affect; Affinity; Bacteria; Bacterial Genes; Binding; Biogenesis; Biological Assay; Biological Models; Biology; Boxing; Carbon; Cell physiology; Code; Congenital Abnormality; Cues; Data; Defect; Double-Stranded RNA; Escherichia coli; Eukaryota; Event; Exhibits; Family; Functional RNA; Gene Expression; Gene Expression Regulation; Gene Targeting; Genetic Epistasis; Genetic Screening; Genetic Transcription; Goals; Growth; Heart; High temperature of physical object; Human; Immunoprecipitation; In Vitro; Infection; Life; Link; Malignant Neoplasms; Mediating; Messenger RNA; Metabolism; Microbial Biofilms; Modeling; Molecular; Mutagenesis; Neurodegenerative Disorders; Organism; Pathway interactions; Phosphorus; Phosphorylation; Physiology; Play; Process; Production; Prokaryotic Cells; Proteins; Proteobacteria; RNA; RNA Decay; RNA Helicase; RNA Splicing; RNA-Binding Proteins; RNA-dependent ATPase; Radioactive; Regulation; Reporting; Ribosomal RNA; Ribosomes; Role; Shock; Signal Transduction; Signal Transduction Pathway; Stimulus; Stress; Structure; System; Techniques; Temperature; Testing; Transcript; Transcriptional Regulation; Translating; Translational Regulation; Translations; Virulence; Virulence Factors; Virus; Work; cell motility; clinically relevant; cold temperature; environmental change; genetic regulatory protein; helicase; human disease; in vivo; mRNA Export; mRNA Stability; mutant; novel; pathogen; pathogenic bacteria; public health relevance; quorum sensing; research study; response

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.

描述（由申请人提供）：该项目的长期目标是加强对基因调控的转录后机制的基本理解。本文研究的碳储存调节系统“Csr”对生物膜的形成、运动性和毒力因子的表达具有深远的影响。变形菌拟议的研究详细说明了一项计划，以调查死亡盒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胁迫期间rRNA成熟，并在RNA周转中辅助次级切割事件。与此相反，本研究探讨了DEAD盒RNA解旋酶的调节作用，其a）影响控制许多细菌毒力因子的全局调节系统的表达，以及B）在与哺乳动物感染相关的温度（37 ℃）下发生。 CsrA是一种RNA结合蛋白，其通过与mRNA的5'非翻译区（5' UTR）结合，从而改变蛋白质翻译和/或RNA周转，转录后调节许多重要的细菌基因和系统。非编码RNA CsrB和CsrC以高亲和力结合多种CsrA蛋白，从而隔离和拮抗该蛋白。这些ncRNA的转录依赖于BarA/UvrY双组分信号转导系统。现有的数据表明，BarA/UvrY电路是中央输入机制传输环境刺激的调节Csr。 转座子诱变筛选鉴定了E.大肠杆菌DEAD盒RNA解旋酶SrmB和DeaD作为CsrB表达的强调节剂。SrmB和DeaD似乎通过BarA/UvrY系统独立地影响CsrB表达。DeaD缺失突变体具有显著降低的UvrY水平，而srmB缺失表现出降低的BarA/UvrY磷酸化，而不影响这些蛋白质的水平。该建议的重点是阐明DeaD和SrmB调节BarA/UvrY的单独途径。 本提案的具体目的是确定（i）DeaD和（ii）SrmB如何独立调节CsrB水平并最终调节CsrA活性。我们将检验DeaD通过促进uvrY翻译来增加uvrY蛋白水平的假设。我们将结合联合收割机在体内磷酸化测定与上位性实验，以进一步表征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。 该提议的目标有三个方面：i）确定DEAD盒RNA解旋酶影响Csr活性的机制，ii）定义一类RNA解旋酶的新的体内靶标，其主要活性被认为是冷休克下的rRNA加工，以及iii）开发用于研究一类重要的生物分子的模型系统，所述生物分子在原核生物、真核生物和病毒中具有不同的功能。