Analysis of E. coli ribonucleases and RNA metabolism

大肠杆菌核糖核酸酶和 RNA 代谢分析

• 批准号: 7760186
• 负责人: Sidney R. Kushner
• 金额: $ 29.21万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-15 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7760186
• 关键词: Accounting; Animal Model; Antibiotic Resistance; Bacteria; Biochemical; Biochemistry; Bioinformatics; Biological; Cells; Collection; Drug Delivery Systems; Endoribonucleases; Enzymes; Escherichia coli; Eukaryota; Exoribonuclease II; Exoribonucleases; Family; Genetic; Genome; Genomics; Knowledge; Metabolism; Modeling; Molecular; Molecular Biology; Mutation; Pancreatic ribonuclease; Pathway interactions; Polyribonucleotide Nucleotidyltransferase; Prevalence; Process; Prokaryotic Cells; Proteins; RNA; RNase P; RNase Z; Reaction; Ribonuclease III; Ribonucleases; Ribosomal RNA; Role; Series; System; Transfer RNA; Work; design; endonuclease; endoribonuclease; genetic analysis; mRNA Decay; mutant; poly A specific exoribonuclease; research study; ribonuclease E; spleen exonuclease; tRNA Precursor

DESCRIPTION (provided by applicant): Although numerous ribonucleases have been identified over the past 40 years, most have been associated primarily with the biological reaction that was used for their identification. Thus an enzyme like RNase P has been assumed to be strictly involved tRNA maturation. Likewise the RNase Z family of enzymes has been assumed to be only involved in tRNA maturation, while RNase III type proteins, at least in prokaryotics are considered rRNA processing enzymes. However, more recent experiments have demonstrated that many of these ribonucleases have multiple functions in the cell. When one carefully looks at what is known about the pathways of rRNA processing, tRNA maturation and mRNA decay, it becomes clear that many of the existing models for these processes are far too simplistic and in some cases probably not correct. In fact, there are many critical gaps in our understanding of these pathways. In addition, not much is known regarding the enzymatic overlap among these important pathways. Accordingly, this application describes a series of experiments that will focus on developing a more complete understanding of post-transcriptional RNA metabolism in the model prokaryote, Escherichia coli. Our approach will be to use a combination of genetics, genomics, molecular biology, and biochemistry to examine the enzymatic steps involved in rRNA maturation, tRNA processing and mRNA decay. We will take advantage of a collection of mutants that we have constructed that contain deficiencies in various combinations of seven endoribonucleases (RNase I, RNase III, RNase E, RNase G, RNase Z, RNase P, and RNase LS) as well as two exoribonucleases (polynucleotide phosphorylase, RNase II). Specific experiments include: 1. Elucidate the multiple pathways involved in tRNA maturation; 2. Analyze the role of RNase III in rRNA processing; 3. Identification and characterization of a 5' -> 3' exoribonuclease; 4. Genetic analysis of the inititation of mRNA decay; and, 5. Determine the primary function for RNase Z in E. coli. With the increasing prevalence of antibiotic resistant bacteria, the need to better understand the overall mechanism of post-transcriptional RNA metabolism is becoming increasingly important. Information gained from this work could be instrumental in the identification of potential new drug targets. Project Narrative Many significant gaps exist in our knowledge of the pathways of rRNA processing, tRNA maturation and mRNA decay. The experiments proposed in this application are designed to develop a more comprehensive overview of these pathways in the model organism Escherichia coli by taking advantage of a unique set of strains carrying various combinations of mutations in nine different ribonucleases, a recently developed tiling chip for the entire E. coli genome, and bioinformatic, molecular biological and biochemical approaches to better characterize the relationships among these known ribonucleases as well as identifying additional enzymes involved in these important post-transcriptional regulatory mechanisms.

描述(申请人提供)：尽管在过去的40年里已经鉴定了许多核糖核酸酶，但大多数主要与用于鉴定它们的生物反应有关。因此，像RNaseP这样的酶被认为严格地参与了tRNA的成熟。同样，RNaseZ家族被认为只参与tRNA的成熟，而RNaseIII型蛋白质，至少在原核生物中被认为是rRNA加工酶。然而，最近的实验表明，这些核糖核酸酶中的许多在细胞中具有多种功能。当人们仔细研究rRNA加工、tRNA成熟和mRNA衰退的途径时，很明显，这些过程的许多现有模型过于简单化，在某些情况下可能不正确。事实上，我们对这些途径的理解存在许多关键差距。此外，关于这些重要途径之间的酶重叠还不是很清楚。因此，本申请描述了一系列实验，这些实验将侧重于更全面地了解模式原核生物大肠杆菌中转录后RNA代谢的情况。我们的方法将是结合遗传学、基因组学、分子生物学和生物化学来研究rRNA成熟、tRNA加工和mRNA衰退所涉及的酶步骤。我们将利用我们构建的一系列突变体，这些突变体包含七种内切核酸酶(RNaseI、RNaseIII、RNaseE、RNaseG、RNaseZ、RNaseP和RNaseLS)以及两种外切核酸酶(多核苷酸磷酸化酶，RNaseII)的各种组合。具体实验包括：1.阐明tRNA成熟所涉及的多种途径；2.分析RNaseIII在rRNA加工中的作用；3.5‘-gt；3’外切核糖核酸酶的鉴定和性质；4.启动mRNA衰退的遗传学分析；以及5.确定RNaseZ在大肠杆菌中的主要功能。随着抗生素耐药细菌的日益流行，更好地了解转录后RNA代谢的整体机制变得越来越重要。从这项工作中获得的信息可能有助于确定潜在的新药靶标。项目描述在我们对rRNA加工、tRNA成熟和信使核糖核酸衰变的途径的了解中存在许多显著的差距。本申请中提出的实验旨在通过利用一组独特的菌株来更全面地概述模式生物大肠杆菌中的这些途径，这些菌株携带九种不同核糖核酸酶的各种突变组合，最近开发的用于整个大肠杆菌基因组的切片芯片，以及生物信息学、分子生物学和生化方法，以更好地表征这些已知核糖核酸酶之间的关系，以及确定参与这些重要的转录后调控机制的其他酶。