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Analysis of E. coli ribonucleases and RNA metabolism

大肠杆菌核糖核酸酶和 RNA 代谢分析

基本信息

批准号：
9980912
负责人：
Sidney R. Kushner
金额：
$ 33.75万
依托单位：
UNIVERSITY OF GEORGIA
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：
2008-03-15 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9980912
关键词：
5&apos -exoribonuclease Animal Model Bacteria Bacterial Antibiotic Resistance Biochemical Bioinformatics Biological Catalytic RNA Categories Cells Data Endoribonucleases Environment Enzymes Escherichia coli Eukaryota Exonuclease Family Gene Expression Gene Expression Regulation Generations Genes Genetic Genetic Transcription Genomics Grant Knowledge Laboratories Link Messenger RNA Modeling Molecular Pathway interactions Phosphodiesterase I Play Polyadenylation Polyribonucleotide Nucleotidyltransferase Post-Transcriptional RNA Processing Prevalence Process Progress Reports Prokaryotic Cells Protein Biosynthesis Proteins RNA RNA Decay RNA Precursors RNA Processing RNA metabolism RNase P RNase Z Reaction Ribonuclease III Ribonucleases Ribosomal RNA Role Series Structure Substrate Specificity Transcript Transfer RNA Transferase Work density endoexonuclease endonuclease experimental study insight mRNA Decay new therapeutic target novel proline-tRNA ribonuclease E tRNA Precursor tRNA adenylyltransferase transcriptome

项目摘要

Project Summary 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 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 P or polynucleotide phosphorylase (PNPase) as the first step in processing tRNA precursors rather than RNase E. Furthermore, we hve shown a link between polyadenylation and functional tRNA levels as well as the existence of a previously unidentified endonuclease. 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 unique bacterial strains, high density tiling microarrays as well as other molecular biological, biochemical and bioinformatic approaches. Specific experiments include: 1. Transcriptome-wide analysis of the initiation of mRNA processing and decay; 2. Detailed analysis of the role of tRNA nucleotidyl transferase in RNA metabolism and identification of structural features for exonuclease-independent tRNA maturation;3. Characterization of YhgF endonuclease activity and the molecular mechanisms of RNase III- independent 30S rRNA processing. 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.

项目概要尽管过去 40 年来已经鉴定出大量核糖核酸酶，但大多数都已被主要与用于识别的生物反应有关。因此 RNase P 等酶被认为严格参与 tRNA 加工。同样的 RNase Z 酶家族也被认为只参与 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，大多数核糖核酸酶细胞内具有多种功能。当人们仔细研究已知的路径时 rRNA 成熟、tRNA 加工和 mRNA 衰变，很明显，许多这些过程的现有模型过于简单，在某些情况下可能不正确。在此外，对于这些重要途径之间的酶促重叠知之甚少。例如，在当前的资助期内，我们已经证明了多个需要 RNase P 或多核苷酸的 tRNA 加工新途径磷酸化酶 (PNPase) 作为处理 tRNA 前体的第一步，而不是 RNase E。此外，我们还展示了多聚腺苷酸化和功能性 tRNA 水平之间的联系因为存在先前未鉴定的核酸内切酶。据此，本申请描述了一系列实验，重点是发展更完整的理解模型原核生物大肠杆菌中转录后 RNA 代谢的研究。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方法。具体实验包括： 1. mRNA加工和衰变起始的全转录组分析； 2. 详细分析tRNA核苷酸转移酶在RNA代谢中的作用鉴定不依赖核酸外切酶的tRNA成熟的结构特征；3． YhgF 核酸内切酶活性表征和 RNase III- 的分子机制独立的30S rRNA处理。随着抗生素耐药性的日益普遍细菌，需要更好地了解转录后RNA的整体机制新陈代谢变得越来越重要。从这项工作中获得的信息可能是有助于识别潜在的新药靶点。