Ribonucleic acid processing activity of Escherichia coli ribonuclease III

Abstract

We have studied the nuclease activities present in preparations of Escherichia coli RNase III and the "sizing factor" responsible for specific processing of several RNA species. RNase III preparations contain three activities: one which solubilizes stable RNA:RNA duplexes; one which solubilizes the RNA of DNA:RNA hybrids; and one which processes the polycistronic mRNA of bacteriophage T7 in a manner identical with sizing factor. We show that the activity against the RNA of DNA:RNA hybrids can be removed, but that the activity which cleaves RNA:RNA duplexes and that responsible for specific processing of phage T7 polycistronic mRNA appear to be identical by several biochemical criteria. In addition, partially purified enzyme fractions from mutants lacking these two activities contain substantial amounts of activity against the RNA of DNA:RNA hybrids. We have also defined several properties of the two activities solubilize RNA:RNA duplexes and RNA of DNA:RNA hybrids. Average oligonucleotide chain length in an exhaustive digest of double-stranded RNA is about 15 bases, while that in a digest of the RNA in DNA:RNA hybrids is less than 10 bases. Direct analysis shows that both activities cleave RNA chains to yield 5'-phosphate and 3'-hydroxyl termini. All four bases can reside at the 5' end of the resulting oligonucleotides, although both activities show a mild preference for certain bases. These results and previous findings allow us to specify the probably size and structure of potential cleavage sites for these enzymes in biological RNA molecules.