title: Bacterial toxin RelE mediates frequent codon-independent mRNA cleavage from the 5' end of coding regions in vivo.
abstract: The enzymatic activity of the RelE bacterial toxin component of the Escherichia coli RelBE toxin-antitoxin system has been extensively studied in vitro and to a lesser extent in vivo. These earlier reports revealed that 1) RelE alone does not exhibit mRNA cleavage activity, 2) RelE mediates mRNA cleavage through its association with the ribosome, 3) RelE-mediated mRNA cleavage occurs at the ribosomal A site and, 4) Cleavage of mRNA by RelE exhibits high codon specificity. More specifically, RelE exhibits a preference for the stop codons UAG and UGA and sense codons CAG and UCG in vitro. In this study, we used a comprehensive primer extension approach to map the frequency and codon specificity of RelE cleavage activity in vivo. We found extensive cleavage at the beginning of the coding region of five transcripts, ompA, lpp, ompF, rpsA, and tufA. We then mapped RelE cleavage sites across one short transcript (lpp) and two long transcripts (ompF and ompA). RelE cut all of these transcripts frequently and efficiently within the first ∼100 codons, only occasionally cut beyond this point, and rarely cut at sites in proximity to the 3' end. Among 196 RelE sites in these five transcripts, there was no preference for CAG or UCG sense codons. In fact, bioinformatic analysis of the RelE cleavage sites failed to identify any sequence preferences. These results suggest a model of RelE function distinct from those proposed previously, because RelE directed frequent codon-independent mRNA cleavage coincident with the commencement of translation elongation.
authors.affiliation: Department of Molecular Genetics, Microbiology, and Immunology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Hurley
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Cruz
authors.forename: Ming
authors.initials: M
authors.lastname: Ouyang
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Woychik
mesh_terms: D024506:5' Flanking Region; D001427:Bacterial Toxins / Q000378:metabolism*; D001665:Binding Sites; D003062:Codon / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D003062:Codon; D029968:Escherichia coli Proteins; D012333:RNA, Messenger