Analysis of E. coli ribonucleases and RNA metabolism

项目摘要 尽管在过去的40年里已经鉴定了许多核糖核酸酶，但大多数核糖核酸酶都是在 主要与用于识别它们的生物反应有关。因而 像RNase P这样的酶被认为严格参与tRNA加工。同样 RNase Z家族的酶也被认为只参与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，大多数这些核糖核酸酶具有 细胞中的多种功能。当一个人仔细观察已知的关于 rRNA成熟，tRNA加工和mRNA衰变，很明显，许多人的基因 这些过程的现有模型过于简单，并且在某些情况下可能是不正确的。在 此外，关于这些重要途径之间的酶重叠，我们知之甚少。 例如，在目前的赠款期间，我们已经证明了存在多个 需要RNase P或多核苷酸的tRNA加工新途径 磷酸化酶（PNase）作为加工tRNA前体的第一步，而不是RNase E。 此外，我们还发现了多聚腺苷酸化和功能性tRNA水平之间的联系 因为存在一种以前未鉴定的核酸内切酶。因此，本申请 描述了一系列的实验，这些实验将专注于开发一个更完整的理解 转录后RNA代谢的模式原核生物，大肠杆菌。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。具体实验 有：1. mRNA加工和降解起始的全转录组分析; 2.详细分析了tRNA核苷酸转移酶在RNA代谢中的作用， 鉴定非核酸外切酶依赖性tRNA成熟的结构特征;3. YhgF内切酶活性的表征和RNase III的分子机制- 独立的30 S rRNA加工。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物靶点。

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.