Mutational analysis of the nuclease domain of Escherichia coli ribonuclease III. Identification of conserved acidic residues that are important for catalytic function in vitro

The ribonuclease III superfamily represents a structurally distinct group of double-strand-specific endonucleases with essential roles in RNA maturation, RNA decay, and gene silencing. Bacterial RNase III orthologs exhibit the simplest structures, with an N-terminal nuclease domain and a C-terminal double-stranded RNA-binding domain (dsRBD), and are active as homodimers. The nuclease domain contains conserved acidic amino acids, which in Escherichia coli RNase III are E38, E41, D45, E65, E100, D114, and E117. On the basis of a previously reported crystal structure of the nuclease domain of Aquifex aeolicus RNase III, the E41, D114, and E117 side chains of E. coli RNase III are expected to be coordinated to a divalent metal ion (Mg(2+) or Mn(2+)). It is shown here that the RNase IIIE41A] and RNase IIID114A] mutants exhibit catalytic activities in vitro in 10 mM Mg(2+) buffer that are comparable to that of the wild-type enzyme. However, at 1 mM Mg(2+), the activities are significantly lower, which suggests a weakened affinity for metal. While RNase IIIE41A] and RNase IIID114A] have K(Mg) values that are approximately 2.8-fold larger than the K(Mg) of RNase III (0.46 mM), the RNase IIIE41A/D114A] double mutant has a K(Mg) of 39 mM, suggesting a redundant function for the two side chains. RNase IIIE38A], RNase IIIE65A], and RNase IIIE100A] also require higher Mg(2+) concentrations for optimal activity, with RNase IIIE100A] exhibiting the largest K(Mg). RNase IIID45A], RNase IIID45E], and RNase IIID45N] exhibit negligible activities, regardless of the Mg(2+) concentration, indicating a stringent functional requirement for an aspartate side chain. RNase IIID45E] activity is partially rescued by Mn(2+). The potential functions of the conserved acidic residues are discussed in the context of the crystallographic data and proposed catalytic mechanisms.