Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain

The ribonuclease III superfamily represents a structurally related group of double-strand (ds) specific endoribonucleases which play key roles in diverse prokaryotic and eukaryotic RNA maturation and degradation pathways. A dsRNA-binding domain (dsRBD) is a conserved feature of the superfamily and is important for substrate recognition. RNase III family members also exhibit a catalytic domain, in part defined by a set of highly conserved amino acids, of which at least one (a glutamic acid) is important for cleavage but not for substrate binding. However, it is not known whether the catalytic domain requires the dsRBD for activity. This report shows that a truncated form of Escherichia coli RNase III lacking the dsRBD (RNase III[Delta dsRBD]) can accurately cleave small processing substrates in vitro. Optimal activity of RNase III[Delta dsRBD] is observed at low salt concentrations (<60 mM Na+), either in the presence of Mg2+ (>25 mM) or Mn2+ (similar to5 mM). At 60 mM Na+ and 5 mM Mn2+ the catalytic efficiency of RNase III[Delta dsRBD] is similar to that of RNase III at physiological salt concentrations and Mg2+. In the presence of Mg2+ RNase III[Delta dsRBD] is less efficient than the wild-type enzyme, due to a higher K-m. Similar to RNase III, RNase III[Delta dsRBD] is inhibited by high concentrations of Mn2+, which is due to metal ion occupancy of an inhibitory site on the enzyme. RNase III[Delta dsRBD] retains strict specificity for dsRNA, as indicated by its inability to cleave (rA)(25), (rU)(25), or (rC)(25). Moreover, dsDNA, ssDNA, or an RNA-DNA hybrid are not cleaved. Low (micromolar) concentrations of ethidium bromide block RNase III[Delta dsRBD] cleavage of substrate, which is similar to the inhibition seen with RNase III and is indicative of an intercalative mode of inhibition. Finally, RNase III[Delta dsRBD] is sensitive to specific Watson-Crick base-pair substitutions which also inhibit RNase III. These findings support an RNase III mechanism of action in which the catalytic domain (i) can function independently of the dsRBD, (ii) is dsRNA-specific, and (iii) participates in cleavage site selection.