Biosynthesis of tRNA in yeast mitochondria. An endonuclease is responsible for the 3'-processing of tRNA precursors.

To study the mechanism involved in the 3'-processing of mitochondrial tRNA precursors, we examined tRNA processing in a reconstituted system with a yeast mitochondrial extract. Two mitochondrial tRNA(Glu) precursors synthesized from SP6 RNA polymerase-directed transcription system were used as substrates. One contained a 214-nucleotide 5' terminus and 115-123-nucleotide 3' trailer. The other had the same sized 3' trailer, but contained a mature 5' terminus. An endonucleolytic activity was identified in a mitochondrial S30 fraction which cleaves the 3' terminus of the latter tRNA precursor precisely at the in vivo CCA addition site. No cleavage of the 5'-extended precursor was observed in vitro. This mitochondrial 3'-processing activity was partially purified using DEAE-CL-6B chromatography. It removes the 3' trailer sequence from the 5'-matured precursor leaving a 3'-hydroxyl group on the processed tRNA and a 5'-phosphate group on the trailer. The resulting tRNA product serves as a substrate for tRNA nucleotidyltransferase which catalyzes the addition of CCA residues to the tRNA to complete its 3' maturation. Thus, yeast mitochondrial 3'-tRNA processing events resemble those found in eucaryotic cytoplasmic/nuclear systems where a single endonucleolytic cleavage is responsible for the formation of the 3' end of the tRNAs. This is in contrast to the multistep 3'-processing events known to occur in procaryotes.