Specific Ribonucleases Involved in Processing of tRNA Precursors of Escherichia coli

Abstract

RNases O and Q, the 2 putative nucleolytic activities which were detected previously in the crude extract from a thermosensitive RNase P mutant (TS241) of E. coli and which functioned in the processing of tRNA precursors in vitro, were partially purified from the 100,000 .times. g supernatant fraction of E. coli Q13. In the course of purification of these enzymes, the total RNA synthesized in the thermosensitive mutant at the restrictive temperature were used as the substrates and the activities were identified from disappearance or alteration of specific tRNA precursor molecules in polyacrylamide gel electrophoresis. The purified RNase O preparation cleaved specifically the multimeric tRNA precursors at the spacer regions. The purified RNase Q preparation removed, in accordance with the definition of this enzyme, extra nucleotides from the 3''-terminal ends of monomeric tRNA precursors. Some properties of these 2 nucleases were investigated. Besides these nucleases, another exonuclease (tentatively designated RNase Y) and RNase P, a well-characterized endonuclease, were also purified. The sequential mode of the processing of tRNA precursors, originally observed in the cleavage reactions with the crude extracts in vitro, was supported by studies with the purified enzyme preparations.