Analysis of E. coli ribonucleases and RNA metabolism

描述（由申请人提供）： 尽管在过去的40年中已经鉴定了许多核糖核酸酶，但大多数主要与用于其鉴定的生物反应有关。因此，像RNase P这样的酶被认为严格参与了tRNA的加工。同样地，RNase Z家族的酶也被认为仅参与tRNA加工，而RNase III型蛋白，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，大多数这些核糖核酸酶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，在某些情况下可能是不正确的。此外，关于这些重要途径之间的酶重叠知之甚少。例如，在目前的资助期内，我们已经证明了存在多种新的tRNA加工途径，这些途径需要RNase E、RNase P或多核苷酸磷酸化酶（PNAPs）作为加工tRNA底物的第一步。因此，本申请描述了一系列实验，这些实验将集中于更全面地了解模型原核生物大肠杆菌中的转录后RNA代谢。我们的方法将使用独特的细菌菌株，高密度平铺微阵列以及其他分子生物学，生物化学和生物信息学方法的组合。具体实验包括：1.确定30 S rRNA加工的分子机制; 2. tRNA加工途径的综合分析;和，3。mRNA加工和衰变起始的全转录组分析。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。

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.