Altering the Divalent Metal Ion Preference of RNase E

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 Mg2+ and Mn2+ will support significant rates of activity in vitro against natural RNAs, with Mn2+ being preferred. Both Mg2+ and Mn2+ also support cleavage of an oligonucleotide substrate with similar kinetic parameters for both ions. Salts of Ni2+ and Zn2+ permitted low levels of activity, while Ca2+, Co3+, Cu2+, and Fe2+ did not. A mutation to one of the residues known to chelate Mg2+, D346C, led to almost complete loss of activity dependent on Mg2+; however, the activity of the mutant enzyme was fully restored by the presence of Mn2+ 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 Mg2+ exclusively in vivo.