An unusual tRNAThr derived from tRNAHis reassigns in yeast mitochondria the CUN codons to threonine

The standard genetic code is used by most living organisms, yet deviations have been observed in many genomes, suggesting that the genetic code has been evolving. In certain yeast mitochondria, CUN codons are reassigned from leucine to threonine, which requires an unusual tRNA(Thr) with an enlarged 8-nt anticodon loop (tRNA(1)(Thr)). To trace its evolutionary origin we performed a comprehensive phylogenetic analysis which revealed that tRNA(1)(Thr) evolved from yeast mitochondrial tRNA His. To understand this tRNA identity change, we performed mutational and biochemical experiments. We show that Saccharomyces cerevisiae mitochondrial threonyl-tRNA synthetase (MST1) could attach threonine to both tRNAThr 1 and the regular tRNA(2)(Thr), but not to the wild-type tRNA His. A loss of the first nucleotide (G(-1)) in tRNA His converts it to a substrate for MST1 with a K-m value (0.7 mu M) comparable to that of tRNA(1)(Thr) (0.3 mu M), and addition of G(-1) to tRNA(1)(Thr) allows efficient histidylation by histidyl-tRNA synthetase. We also show that MST1 from Candida albicans, a yeast in which CUN codons remain assigned to leucine, could not threonylate tRNA(1)(Thr) , suggesting that MST1 has coevolved with tRNA(1)(Thr). Our work provides the first clear example of a recent recoding event caused by alloacceptor tRNA gene recruitment.