E. coli RNase I exhibits a strong Ca2+-dependent inherent double-stranded RNase activity

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Escherichia coli RNase I was traditionally characterized as a single-strand specific RNA endonuclease.
• Its activity was considered largely sequence-independent on single-stranded RNA (ssRNA).

Purpose of the Study:

• To investigate the role of calcium (Ca2+) in modulating RNase I activity.
• To explore RNase I's substrate specificity beyond ssRNA.
• To identify potential applications of Ca2+-dependent RNase I activity.

Main Methods:

• Enzyme activity assays using various RNA substrates (ssRNA, dsRNA, DNA:RNA hybrids).
• Site-directed mutagenesis to investigate the Ca2+ binding site.
• Comparison of wild-type and mutant RNase I activities in the presence and absence of Ca2+.

Main Results:

• RNase I demonstrated significant Ca2+-dependent activity on double-stranded RNA (dsRNA) and DNA:RNA hybrids.
• Ca2+ did not affect RNase I's activity on ssRNA, indicating specific modulation.
• Mutation of a Ca2+ binding site resulted in a gain-of-function mutant with high dsRNA activity, independent of Ca2+ stimulation.

Conclusions:

• Native RNase I possesses bound Ca2+, enabling it to degrade both ssRNA and dsRNA, thus exhibiting broader substrate specificity than previously known.
• The Ca2+-dependent dsRNase activity, distinct from its ssRNase activity, presents a potential tool for applied molecular biology.