Analysis of E. coli ribonucleases and RNA metabolism

描述(由申请人提供)： 虽然在过去的40年里已经发现了许多核糖核酸酶，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参与了tRNA的加工。同样，RNaseZ家族也被认为只参与tRNA的加工，而RNaseIII型蛋白质，至少在原核生物中被认为是rRNA成熟酶。然而，最近的实验表明，这些核糖核酸酶中的大多数在细胞中具有多种功能。当人们仔细研究rRNA成熟、tRNA加工和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，在某些情况下可能是错误的。此外，关于这些重要途径之间的酶重叠还不是很清楚。例如，在当前的授权期内，我们已经证明了存在多种新的tRNA处理途径，这些途径需要RNaseE、RNaseP或多核苷酸磷酸化酶(PNPase)作为处理tRNA前体的第一步。相应地，本申请描述了一系列实验，这些实验将集中在发展对模式原核生物大肠杆菌转录后RNA代谢的更完整的了解。我们的方法将是使用独特的细菌菌株、高密度瓷砖微阵列以及其他分子生物学、生化和生物信息学方法的组合。具体实验包括：1.确定30SRRNA加工的分子机制(S)；2.综合分析tRNA加工途径；3.转录组分析mRNA加工和衰退的起始。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。 公共卫生相关性： 项目描述在我们对rRNA成熟、tRNA加工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，携带各种核糖核酸酶和RNA解旋酶的各种突变组合，最近针对整个大肠杆菌基因组的高密度平铺微阵列，以及生物信息学、分子生物学和生化方法，对模式生物大肠杆菌中的这些途径进行更全面的概述。更好地了解转录后RNA代谢将为细菌生命周期提供重要的新见解。

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 processing. Likewise the RNase Z family of enzymes has also been thought to be only involved in tRNA processing, while RNase III type proteins, at least in prokaryotes are considered rRNA maturation enzymes. However, more recent experiments have demonstrated that most of these ribonucleases have multiple functions in the cell. When one carefully looks at what is known about the pathways of rRNA maturation, tRNA processing and mRNA decay, it becomes clear that many of the existing models for these processes are far too simplistic and in some cases probably incorrect. In addition, not much is known regarding the enzymatic overlap among these important pathways. For example, during the current grant period, we have demonstrated the existence of multiple new pathways for tRNA processing that require either RNase E, RNase P or polynucleotide phosphorylase (PNPase) as the first step in processing tRNA precursorsAccordingly, 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 unique bacterial strains, high density tiling microarrays as well as other molecular biological, biochemical and bioinformatic approaches. Specific experiments include: 1. Determine the molecular mechanism(s) of 30S rRNA processing; 2. Comprehensive analysis of tRNA processing pathways; and, 3. Transcriptome-wide analysis of the initiation of mRNA processing and decay. 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. PUBLIC HEALTH RELEVANCE: Project Narrative Many significant gaps exist in our knowledge of the pathways of rRNA maturation, tRNA processing 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 various ribonucleases and RNA helicases, a recently high density tiling microarray for the entire E. coli genome, and bioinformatic, molecular biological and biochemical approaches. A better understanding of post-transcriptional RNA metabolism will provide important new insights into the bacterial life cycle.