Pre-tRNA 3′-processing in <em>Saccharomyces cerevisiae</em>.

We investigated ribonucleases from Saccharomyces cerevisiae which are active in pre-tRNA 3&#39;-pro cessing in vitro. Two pre-tRNA 3&#39;-exonucleases with molecular masses of 33 and 60 kDa, two pre-tRNA -endonucleases with molecular masses of 45 kDa/60 kDa and 55 kDa and a 70-kDa 3&#39;-pre-tRNase were paraph from yeast whole cell extracts by several successive chromatographic purification steps. The purified exonucleases are non-processive 3&#39;-exonucleases that catalyze the exonucleolytic processing of 3&#39;-trailer sequences of pre-tRNAs to produce mature tRNAs. The 45-kDa/60-kDa 3&#39;-endonuclease is tRNA-specific and catalyzes the processing of tRNAs in a single endonucleolytic steps. Two isoenzymes of this activity (p45 and p60) were identified by chromatography. The second endonuclease, p55, is dependent on monovalent ions and cleaves about three nucleotides downstream the mature 3&#39;-end. All of the purified 3&#39;-pre-tRNases accept homologous as well as heterologous pre-tRNA substrates. Pre-tRNAs carrying a 5&#39;-leader are processed with almost the same efficiency as those lacking this 5&#39;-leader. Mature tRNA carrying 3&#39;-sequence and tRNA pseudogene products carrying mutations in the mature domain are processed by the 3&#39;-exonucleases, not by the 3&#39;-endonucleases. The specific endonuclease p45/p60 discriminates between UUUOH as a 3&#39;-flank, which is cleaved, and the CCA 3&#39;-end of mature tRNAs, which is not cleaved. This study suggests that several 3&#39;-pre-tRNases are active on tRNA precursors in vitro and might therefore participate in pre-tRNA 3&#39;-processing in yeast, partly in cooperative manner. [ABSTRACT FROM AUTHOR]