Analysis of E. coli ribonucleases and RNA metabolism

描述（由申请方提供）：尽管在过去40年中已鉴定出许多核糖核酸酶，但大多数主要与用于其鉴定的生物反应相关。因此，像RNase P这样的酶被认为严格参与了tRNA的成熟。类似地，RNase Z家族的酶被认为仅参与tRNA成熟，而RNase III型蛋白，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，许多这些核糖核酸酶在细胞中具有多种功能。当我们仔细研究rRNA加工、tRNA成熟和mRNA衰变的已知途径时，很明显，这些过程的许多现有模型过于简单，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知之甚少。因此，本申请描述了一系列的实验，将集中在开发一个更完整的理解在模型原核生物，大肠杆菌的转录后RNA代谢。我们的方法将是使用遗传学，基因组学，分子生物学和生物化学的组合，以检查参与rRNA成熟，tRNA加工和mRNA衰变的酶步骤。我们将利用我们已经构建的突变体的集合，这些突变体在七种核糖核酸内切酶（RNA酶I、RNA酶III、RNA酶E、RNA酶G、RNA酶Z、RNA酶P和RNA酶LS）以及两种核糖核酸外切酶（多核苷酸磷酸化酶，RNA酶II）的各种组合中含有缺陷。具体实验包括：1.阐明tRNA成熟过程中涉及的多种途径; 2.分析RNase III在rRNA加工中的作用; 3. 5' -> 3'核糖核酸外切酶的鉴定和表征; 4. mRNA衰变起始的遗传分析;和，5。确定RNase Z在大肠杆菌中的主要功能。杆菌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物靶点。在我们对rRNA加工、tRNA成熟和mRNA降解途径的认识中存在许多重大空白。本申请中提出的实验旨在通过利用一组独特的菌株（在9种不同的核糖核酸酶中携带各种突变组合），对模式生物大肠杆菌中的这些途径进行更全面的概述，最近开发的用于整个大肠杆菌的平铺芯片。大肠杆菌基因组，生物信息学，分子生物学和生物化学的方法，以更好地表征这些已知的核糖核酸酶之间的关系，以及确定其他酶参与这些重要的转录后调控机制。

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.