E. coli RNase I exhibits a strong Ca(2+)-dependent inherent double-stranded RNase activity

Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca(2+))-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca(2+)-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca(2+) does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca(2+) in the modulation of RNase I activity. Mutation of a previously overlooked Ca(2+) binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca(2+), allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca(2+)-dependency of RNase I may be useful as a tool in applied molecular biology.