Comparative Analysis of RNA Helicases in E. coli

大肠杆菌 RNA 解旋酶的比较分析

基本信息

批准号：
7769554
负责人：
Chaitanya Jain
金额：
$ 29.26万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
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/7769554
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&apos 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

项目摘要

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）：DEAD-box RNA解旋酶是一类多功能的保守蛋白，其特征在于它们能够解开RNA双链体，据信它们在调节RNA功能中发挥重要作用。然而，许多DEAD-box蛋白的研究很少，因此它们的细胞功能尚不清楚。如果没有这些知识，对 RNA 代谢的详细了解仍然是不完整的。我们建议研究两种大肠杆菌 DEAD-box RNA 解旋酶 SrmB 和 DeaD，它们与 RNA 代谢的不同方面有关。我们的初步数据表明：(i) SrmB 和 DeaD 各自促进非进行性外切核糖核酸酶、多核苷酸磷酸化酶 (PNPase)，以在体内消化结构化 RNA； (ii) 任何一个因素的缺失都会导致 8% 的大肠杆菌转录物的差异调节，大概是通过对 RNA 构象和周转的影响； (iii) srmB和deaD菌株的冷敏感生长表型可以通过过表达核糖体RNA加工和修饰因子来抑制； (iv) 大肠杆菌 DEAD-box 解旋酶刺激聚腺苷酸聚合酶 (PAP) 的功能，这种酶可使细胞转录物聚腺苷酸化并靶向其降解。因此，这些蛋白质中的每一种在 RNA 周转中都具有多种作用。结合遗传和生化方法，拟议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，以确定通过 SrmB 或 DeaD 刺激 PNPase 功能进行更新的细胞转录本。我们还将对导致这些特性的 RNA 决定簇进行生化表征。其次，我们将鉴定与细胞中的 SrmB 或 DeaD 直接相互作用的转录本，并研究这些因子差异转录调控的机制。第三，我们将研究抑制由于核糖体RNA加工因子过度表达而导致的srmB和deaD冷敏感生长缺陷的基础。第四，我们将分析 DEAD-box 解旋酶刺激 PAP 的机制和后果。总体而言，这些研究应该为靶标选择的基础、与 RNA 加工酶的功能相互作用以及两种重要的 DEAD-box 蛋白的细胞功能提供见解。这些发现应该与尚未详细表征的 DEAD-box 蛋白广泛相关。项目叙述通过更好地了解 RNA 代谢，拟议的 DEAD-box RNA 解旋酶研究将更好地了解由 RNA 功能缺陷引起的疾病。对此类 RNA 解旋酶的深入了解也可能有助于设计针对病原生物中保守的 DEAD-box 结构域的新型抗生素。