Purification and Properties of Ribonuclease III from Escherichia coli

Abstract

Abstract A nuclease with specificity for double-stranded RNA (RNase III) has been found in extracts of Escherichia coli. It remains within osmotically shocked cells attached to the ribosomes. It sediments with the ribosomes in less than 0.20 m NH4Cl, but is detached at higher concentrations. A purification is described which utilizes this property. The free enzyme, after diethylaminoethyl Sephadex and carboxymethyl Sephadex chromatography, shows an absolute specificity for polymers containing double helical polyribonucleotide regions—other polymers (single- and double-stranded DNAs, single-stranded RNAs) are not digested, nor do they inhibit the digestion of double-stranded RNAs when present in excess. RNase III shows an absolute requirement for divalent cations, including Mg++ and Mn++, and for monovalent cations, including NH4+, K+, and Na+. The optimal pH range for the reaction is from pH 7.6 to pH 9.75. RNase III is inactivated by exposure to many substances at monovalent cation concentrations below 0.2 m. The enzyme is not a factor required for protein synthesis in vitro. Its mode of action appears to be endonucleolytic.