Specific ribonucleases involved in processing of tRNA precursors of Escherichia coli. Partial purification and some properties.
Ribonucleases O and Q, the two putative nucleolytic activities which we detected previously in the crude extract from a thermosensitive ribonuclease P mutant (TS241) of Escherichia coli and which were shown to function in the processing of tRNA precursors in vitro, were partially purified from the 1000000 x g supernatant fraction of E. coli Q13. In the course of purification of these enzymes, the total RNAs 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 ribonuclease O preparation cleaved specifically the multimeric tRNA precursors at the spacer regions. The purified ribonuclease 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 two nucleases were investigated. In addition to these nucleases, another exonuclease (tentatively designated ribonuclease Y) and ribonuclease 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.
D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D012343:RNA, Transfer / Q000096:biosynthesis*; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature
D012343:RNA, Transfer; D012260:Ribonucleases