Comparative Analysis of RNA Helicases in E. coli

大肠杆菌 RNA 解旋酶的比较分析

• 批准号: 7587468
• 负责人: Chaitanya Jain
• 金额: $ 29.5万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7587468
• 关键词: Affinity; Affinity Chromatography; Animal Model; Antibiotics; Binding Sites; Biochemical; Biological Assay; Boxing; Cell physiology; Cells; Data; Defect; Digestion; Disease; Environment; Enzymes; Escherichia coli; Exhibits; Future; Gene Expression; Genetic; Genomics; Goals; Growth; Knowledge; Laboratories; Laboratory Study; Messenger RNA; Metabolism; Modification; Molecular; Molecular Conformation; Organism; Phenotype; Play; Poly A; Poly(A) Tail; Polynucleotide Adenylyltransferase; Polyribonucleotide Nucleotidyltransferase; Positioning Attribute; Process; Property; Proteins; RNA; RNA 3' End Processing; RNA Conformation; RNA Helicase; RNA Processing; Regulation; Research; Ribonucleases; Ribosomal RNA; Ribosomes; Role; Site; Structure; Surveys; Testing; Transcript; base; comparative; design; exoribonuclease T; genome wide association study; genome-wide; helicase; in vitro activity; in vivo; insight; novel

DESCRIPTION (provided by applicant): DEAD-box RNA helicases are a versatile class of conserved proteins characterized by their ability to unwind RNA duplexes, which are believed to play important roles in regulating RNA function. However, many DEAD- box proteins have been poorly studied and hence their cellular functions are unknown. Without this knowledge, a detailed understanding of RNA metabolism remains incomplete. We propose to study two E. coli DEAD-box RNA helicases, SrmB and DeaD, which have been implicated in different aspects of RNA metabolism. Our preliminary data indicate the following: (i) SrmB and DeaD each promote a non-processive exo-ribonuclease, polynucleotide phosphorylase (PNPase), to digest structured RNA in vivo; (ii) the absence of either factor results in differential regulation of 8% of E. coli transcripts, presumably through an effect on RNA conformation and turnover; (iii) the cold-sensitive growth phenotype of srmB and deaD strains can be suppressed by over-expressing ribosomal RNA processing and modification factors; (iv) E. coli DEAD-box helicases stimulate the function of poly(A) polymerase (PAP), an enzyme that polyadenylates cellular transcripts and targets them for degradation. Therefore, each of these proteins has multiple roles in RNA turnover. Using a combination of genetic and biochemical approaches, the focus of the proposed studies will be to define these functions in detail. First, we will conduct a genome-wide survey to identify cellular transcripts that undergo turnover via SrmB or DeaD stimulation of PNPase function. We will also biochemically characterize the RNA determinants that are responsible for these properties. Second, we will identify transcripts that directly interact with SrmB or DeaD in the cell, and investigate the mechanism of differential transcript regulation by these factors. Third, we will investigate the basis for suppression of the srmB and deaD cold-sensitive growth defect due to the over-expression of ribosomal RNA processing factors. Fourth, we will analyze the mechanism and consequences of PAP stimulation by DEAD-box helicases. Overall, these studies should provide insights into the basis of target selection, functional interactions with RNA processing enzymes and the cellular functions of two important DEAD-box proteins. Such findings should be broadly relevant to DEAD-box proteins that have yet to be characterized in detail. PROJECT NARRATIVE By providing a greater understanding of RNA metabolism, the proposed studies on DEAD-box RNA helicases will provide better insights into diseases that are due to defects in RNA function. Advanced knowledge of such RNA helicases could also be helpful to design novel antibiotics that target conserved DEAD-box domains in pathogenic organisms.

描述（由申请人提供）：死盒RNA解旋酶是一类多功能的保守蛋白，其特征是它们放弃RNA双链体的能力，据信它们在调节RNA功能中起重要作用。但是，许多死盒蛋白的研究很少，因此它们的细胞功能尚不清楚。没有这些知识，对RNA代谢的详细理解仍然不完整。我们建议研究两个大肠杆菌死盒RNA解旋酶，SRMB和死亡，这与RNA代谢的不同方面有关。我们的初步数据表明：（i）SRMB和死亡每个人都会促进非过程的外核核酸酶，多核苷酸磷酸酶（PNPase），以在体内消化结构化的RNA； （ii）没有任何一个因子会导致8％的大肠杆菌转录本的差异调节，大概是通过对RNA构象和周转的影响； （iii）通过表达过度表达的核糖体RNA加工和修饰因子可以抑制SRMB和死亡菌株的冷敏感生长表型； （iv）大肠杆菌死盒解旋酶刺激聚（A）聚合酶（PAP）的功能，该酶是聚腺苷酸化的酶，可聚腺苷酸化，并将其靶向它们以降解。因此，这些蛋白质中的每一个在RNA更新中都有多个作用。结合遗传和生化方法，拟议的研究的重点将是详细定义这些功能。首先，我们将进行全基因组调查，以识别通过SRMB或PNPase功能死亡刺激进行营业的细胞转录本。我们还将在生化上表征负责这些特性的RNA决定因素。其次，我们将确定直接与SRMB或细胞中死亡相互作用的成绩单，并通过这些因素调查差异转录的机理。第三，由于核糖体RNA加工因子过表达，我们将研究抑制SRMB和死亡敏感的生长缺陷的基础。第四，我们将分析死盒解旋酶的PAP刺激的机制和后果。总体而言，这些研究应提供有关目标选择，与RNA加工酶的功能相互作用以及两个重要的死盒蛋白的细胞功能的见解。这些发现应与尚未详细表征的死盒蛋白广泛相关。项目叙述 通过对RNA代谢有更深入的了解，对死盒RNA解旋酶的拟议研究将为您提供有关RNA功能缺陷引起的疾病的更好见解。此类RNA解旋酶的先进知识也可能有助于设计靶向致病生物中保守的死盒结构域的新型抗生素。