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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM081554-04
Application #
8018082
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Tompkins, Laurie
Project Start
2008-03-15
Project End
2012-07-31
Budget Start
2011-02-01
Budget End
2012-07-31
Support Year
4
Fiscal Year
2011
Total Cost
$289,130
Indirect Cost
Name
University of Georgia
Department
Genetics
Type
Schools of Arts and Sciences
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
Mohanty, Bijoy K; Kushner, Sidney R (2018) Enzymes Involved in Posttranscriptional RNA Metabolism in Gram-Negative Bacteria. Microbiol Spectr 6:
Bowden, Katherine E; Wiese, Nicholas S; Perwez, Tariq et al. (2017) The rph-1-Encoded Truncated RNase PH Protein Inhibits RNase P Maturation of Pre-tRNAs with Short Leader Sequences in the Absence of RppH. J Bacteriol 199:
Mildenhall, Kristen B; Wiese, Nicholas; Chung, Daewhan et al. (2016) RNase E-based degradosome modulates polyadenylation of mRNAs after Rho-independent transcription terminators in Escherichia coli. Mol Microbiol 101:645-55
Agrawal, Ankit; Mohanty, Bijoy K; Kushner, Sidney R (2014) Processing of the seven valine tRNAs in Escherichia coli involves novel features of RNase P. Nucleic Acids Res 42:11166-79
Mohanty, Bijoy K; Kushner, Sidney R (2013) Deregulation of poly(A) polymerase I in Escherichia coli inhibits protein synthesis and leads to cell death. Nucleic Acids Res 41:1757-66
Stead, Mark B; Agrawal, Ankit; Bowden, Katherine E et al. (2012) RNAsnap™: a rapid, quantitative and inexpensive, method for isolating total RNA from bacteria. Nucleic Acids Res 40:e156
Mohanty, Bijoy K; Maples, Valerie F; Kushner, Sidney R (2012) Polyadenylation helps regulate functional tRNA levels in Escherichia coli. Nucleic Acids Res 40:4589-603
Mohanty, Bijoy K; Kushner, Sidney R (2011) Bacterial/archaeal/organellar polyadenylation. Wiley Interdiscip Rev RNA 2:256-76
Stead, Mark B; Marshburn, Sarah; Mohanty, Bijoy K et al. (2011) Analysis of Escherichia coli RNase E and RNase III activity in vivo using tiling microarrays. Nucleic Acids Res 39:3188-203
Mohanty, Bijoy K; Kushner, Sidney R (2010) Processing of the Escherichia coli leuX tRNA transcript, encoding tRNA(Leu5), requires either the 3'-->5' exoribonuclease polynucleotide phosphorylase or RNase P to remove the Rho-independent transcription terminator. Nucleic Acids Res 38:597-607

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