Purification and properties of a specific Escherichia coli ribonuclease which cleaves a tyrosine transfer ribonucleic acid presursor.

Precursor molecules of Escherichia coli wild type and mutant tyrosine tRNA’s contain at both their 5’ and 3’ termini extra nucleotides in addition to those of the mature tRNA molecule. The early steps of processing these precursor molecules must involve specific ribonuclease cleavage. We report the isolation from E. coli extracts of the specific endonucleolytic RNase which cleaves only a single phosphodiester bond of the 129 nucleotide tyrosine tRNA precursor molecule. This cleavage removes all extra nucleotides present at the 5’ terminus of the precursor as a 41 nucleotide fragment, exposing the 5’ end of the mature tRNA. After sufficient purification, this activity has no effect upon the extra nucleotides at the 3’ end of the tRNA precursor. Therefore processing of the two ends of this molecule must be carried out by different enzymatic activities. This novel RNase activity, which we have called RNase P, has been purified by washing ribosomes with 0.2 M NH&l, followed by ammonium sulfate fractionation and chromatography on DEAE-Sephadex and phosphocellulose. At this stage it shows no evidence of other E. coli RNase activities. RNase P requires both monovalent and divalent cations for optimal activity, and has a pH optimum of 8.0. In the course of purifying RNase P, we have discovered in other subcellular fractions of E. coli RNase activities potentially responsible for additional steps of precursor tRNA processing.