title: Bacterial toxin RelE: a highly efficient ribonuclease with exquisite substrate specificity using atypical catalytic residues.
abstract: The toxin RelE is a ribosome-dependent endoribonuclease implicated in diverse cellular processes, including persistence. During amino acid starvation, RelE inhibits translation by cleaving ribosomal A-site mRNA. Although RelE is structurally similar to other microbial endoribonucleases, the active-site amino acid composition differs substantially and lacks obvious candidates for general acid-base functionality. Highly conserved RelE residues (Lys52, Lys54, Arg61, Arg81, and Tyr87) surround the mRNA scissile phosphate, and specific 16S rRNA contacts further contribute to substrate positioning. We used a single-turnover kinetic assay to evaluate the catalytic importance of individual residues in the RelE active site. Within the context of the ribosome, RelE rapidly cleaves A-site mRNA at a rate similar to those of traditional ribonucleases. Single-turnover rate constants decreased between 10(2)- and 10(6)-fold for the RelE active-site mutants of Lys52, Lys54, Arg61, and Arg81. RelE may principally promote catalysis via transition-state charge stabilization and leaving-group protonation, in addition to achieving in-line substrate positioning in cooperation with the ribosome. This kinetic analysis complements structural information to provide a foundation for understanding the molecular mechanism of this atypical endoribonuclease.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06511, United States.
authors.forename: Meghan A
authors.initials: MA
authors.lastname: Griffin
authors.forename: Jared H
authors.initials: JH
authors.lastname: Davis
authors.forename: Scott A
authors.initials: SA
authors.lastname: Strobel
mesh_terms: D019943:Amino Acid Substitution / Q000502:physiology; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D012333:RNA, Messenger; C465566:RelE protein, E coli; D012260:Ribonucleases