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.
Highlights
MethodsAssay of Ribonuclease III-The standard reaction mixture for assaying RNase III contains, in a final volume of from 0.05 to 0.5 ml, 0.10 M NH&l, 0.01 M magnesium acetate, 0.02 MTris-HCl (final pH, 7.6), substrate (in concentrations usually in the range near 50 mpmoles of polymer phosphorus per ml), and enzyme
RNase III shows an absolute requirement for divalent cations, including Mgf+ and Mn+f, and for monovalent cations, including NHd+, K+, and Na+
We report here the intracellular localiiation, a partial purification, and the characterization of an enzyme from E. coli which degrades double-stranded RNA
Summary
Assay of Ribonuclease III-The standard reaction mixture for assaying RNase III contains, in a final volume of from 0.05 to 0.5 ml, 0.10 M NH&l, 0.01 M magnesium acetate, 0.02 MTris-HCl (final pH, 7.6), substrate (in concentrations usually in the range near 50 mpmoles of polymer phosphorus per ml), and enzyme. E,ovine serum albumin carrier (0.2 mg) is added, and the precipitate which forms is collected and washed with 5% TCA on Whatman GF/A glass fiber filter pads. The pads are dried and radioactivity is determined in 5 ml of toluene-based scintillation fluid as described below. Radioactivity was determined in a Nuclear-Chicago liquid scintillation counter, model 6804, with a toluene-based scintillation fluid containing 2,5-diphenyloxazole (PPO; 6 g per liter) and 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benzene TCA-precipitable radioactivity was measured by precipitation in 5”/, TCA with the addition of 0.2 mg of bovine serum albumin (Fraction V from Armour Pharmaceutical Company, Kankakee, Illinois) as carrier, followed by filtration through Whatman GF/A glass fiber filter pads, washing with 100 ml of 5% TCA, and a lo-min drying period under an infrared lamp
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