Analysis of E. coli ribonucleases and RNA metabolism

项目摘要 尽管在过去的40年里已经发现了许多核糖核酸酶，但大多数都是 主要与用于鉴定它们的生物反应有关。因此，一个 像RNaseP这样的酶被认为严格参与了tRNA的加工。同样的， 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 在单元格中具有多种功能。当一个人仔细地看一看关于 RRNA成熟、tRNA加工和mRNA衰退，很明显，许多 这些过程的现有模型过于简单化，在某些情况下可能是不正确的。在……里面 此外，人们对这些重要途径之间的酶重叠知之甚少。 例如，在目前的赠款期间，我们已经证明存在多个 需要RNaseP或多核苷酸的tRNA加工的新途径 磷酸酶(PNPase)作为处理tRNA前体的第一步，而不是RNase E。 此外，我们还证明了多聚腺苷酸化与功能性trna水平之间的联系。 因为存在一种以前未知的核酸内切酶。因此，此应用程序 描述了一系列实验，这些实验将侧重于发展更完整的理解 在模式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、高密度瓷砖微阵列以及 其他分子生物学、生化和生物信息学方法。具体实验 包括：1.转录组分析mRNA的起始加工和衰变； 2.详细分析了tRNA核苷酸转移酶在RNA代谢和 鉴定非核酸外切酶依赖的tRNA成熟的结构特征； YhgF核酸内切酶活性鉴定及RNaseIII- 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 新陈代谢正变得越来越重要。从这项工作中获得的信息可能是 有助于确定潜在的新药物靶标。

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.