title: Altering the divalent metal ion preference of RNase E.
abstract: RNase E is a major intracellular endoribonuclease in many bacteria and participates in most aspects of RNA processing and degradation. RNase E requires a divalent metal ion for its activity. We show that only Mg(2+) and Mn(2+) will support significant rates of activity in vitro against natural RNAs, with Mn(2+) being preferred. Both Mg(2+) and Mn(2+) also support cleavage of an oligonucleotide substrate with similar kinetic parameters for both ions. Salts of Ni(2+) and Zn(2+) permitted low levels of activity, while Ca(2+), Co(3+), Cu(2+), and Fe(2+) did not. A mutation to one of the residues known to chelate Mg(2+), D346C, led to almost complete loss of activity dependent on Mg(2+); however, the activity of the mutant enzyme was fully restored by the presence of Mn(2+) with kinetic parameters fully equivalent to those of wild-type enzyme. A similar mutation to the other chelating residue, D303C, resulted in nearly full loss of activity regardless of metal ion. The properties of RNase E D346C enabled a test of the ionic requirements of RNase E in vivo. Plasmid shuffling experiments showed that both rneD303C (i.e., the rne gene encoding a D-to-C change at position 303) and rneD346C were inviable whether or not the selection medium was supplied with MnSO4, implying that RNase E relies on Mg(2+) exclusively in vivo.
authors.affiliation: Department of Biochemistry and Molecular Biology, Life Sciences Centre, The University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Katharine J
authors.initials: KJ
authors.lastname: Thompson
authors.affiliation: Department of Biochemistry and Molecular Biology, Life Sciences Centre, The University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Jeff
authors.initials: J
authors.lastname: Zong
authors.affiliation: Department of Biochemistry and Molecular Biology, Life Sciences Centre, The University of British Columbia, Vancouver, British Columbia, Canada george.mackie@ubc.ca.
authors.forename: George A
authors.initials: GA
authors.lastname: Mackie
mesh_terms: D019943:Amino Acid Substitution; D002413:Cations, Divalent / Q000378:metabolism*; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D020125:Mutation, Missense
chemical_list: D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D008670:Metals; D004722:Endoribonucleases; C022537:ribonuclease E