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 maturation. Likewise the RNase Z family of enzymes has been assumed to be only involved in tRNA maturation, while RNase III type proteins, at least in prokaryotics are considered rRNA processing enzymes. However, more recent experiments have demonstrated that many of these ribonucleases have multiple functions in the cell. When one carefully looks at what is known about the pathways of rRNA processing, tRNA maturation and mRNA decay, it becomes clear that many of the existing models for these processes are far too simplistic and in some cases probably not correct. In fact, there are many critical gaps in our understanding of these pathways. In addition, not much is known regarding the enzymatic overlap among these important pathways. 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 genetics, genomics, molecular biology, and biochemistry to examine the enzymatic steps involved in rRNA maturation, tRNA processing and mRNA decay. We will take advantage of a collection of mutants that we have constructed that contain deficiencies in various combinations of seven endoribonucleases (RNase I, RNase III, RNase E, RNase G, RNase Z, RNase P, and RNase LS) as well as two exoribonucleases (polynucleotide phosphorylase, RNase II). Specific experiments include: 1. Elucidate the multiple pathways involved in tRNA maturation;2. Analyze the role of RNase III in rRNA processing;3. Identification and characterization of a 5'->3'exoribonuclease;4. Genetic analysis of the inititation of mRNA decay;and, 5. Determine the primary function for RNase Z in E. coli. 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. Project Narrative Many significant gaps exist in our knowledge of the pathways of rRNA processing, tRNA maturation 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 nine different ribonucleases, a recently developed tiling chip for the entire E. coli genome, and bioinformatic, molecular biological and biochemical approaches to better characterize the relationships among these known ribonucleases as well as identifying additional enzymes involved in these important post-transcriptional regulatory mechanisms.
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