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

Abstract

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 ( ). To trace its evolutionary origin we performed a comprehensive phylogenetic analysis which revealed that 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 and the regular , 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 μM) comparable to that of (0.3 μM), and addition of G(-1) to 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 , suggesting that MST1 has coevolved with . Our work provides the first clear example of a recent recoding event caused by alloacceptor tRNA gene recruitment.

Figure 1.

Nucleotide sequences of S. cerevisiae mitochondrial tRNAs. (A) with an 8-nt anticodon loop and a UAG anticodon. (B) with a canonical UGU anticodon. (C) tRNA^His with a G₋₁ (circled, a major anti-determinant for ThrRS). The primary sequences of and tRNA^His are 72% identical (shaded).

Figure 2.

Phylogeny of yeast mitochondrial tRNAs. The phylogenetic analysis with PhyloBayes (default model parameters) contained all tRNA sequences from the species shown in Figure 3. Only the section of the tRNA phylogeny covering the and tRNA^His clusters are shown (marked red and blue, respectively), confirming monophyly of and a sister group relationship to tRNA^His. The posterior probability support for the two tRNA groups is 1.0 (note that phylogenetic analysis with tRNA sequences depends on only few informative nucleotide positions, which does not allow to resolve the branching order within these groups). Removal of the anticodon sequence positions from the dataset did not change clustering into the two tRNA groups, nor did an analysis of the same datasets with maximum likelihood and the GTR model.

Figure 3.

Phylogeny of yeast species based on concatenated mtDNA-encoded protein sequences. The phylogenetic analysis with PhyloBayes and the CAT model is based on 13 mtDNA encoded proteins. All divergence points are supported by posterior probability values of 1.0, except where indicated. The red arrow points to the concomitant loss of all seven nad genes and the start of mitochondrial codon reassignments, including AUA methionine, CUN threonine. Species shown in black possess mitochondrial (but not ), and in these organisms CUN codons are assigned to Leu. The yeast species marked red, such as K. thermotolerans, have lost mitochondrial and obtained that decodes CUN codons as Thr. K. lactis is marked magenta as it has no CUN codons and no corresponding tRNA with a UAG anticodon. A. gossypii (marked blue) contains a tRNA species with a UAG anticodon, yet its identity of this tRNA and the amino acid reading CUN codons in A. gossypii remain obscure (to be discussed by BFL in Organelle Genetics: Evolution of Organelle Genomes and Gene Expression, Springer 2011).

Figure 4.

Aminoacylation by S. cerevisiae MST1 and HisRS. (A) Leucylation of (3 µM) and (3 µM) by ScmtLeuRS (0.3 µM). (B) Threonylation of (3 µM), (3 µM) and tRNA^His variants (3 µM) by ScMST1 (0.3 µM). (C) Histidylation of (3 µM) and tRNA^His variants (3 µM) by ScmtHisRS (0.3 µM).

Figure 5.

Sequence alignment of mitochondrial and tRNA^His. Three major differences between and tRNA^His sequences are indicated by boxes. Cc, Candida castellii; Cg, Candida glabrata; Kt, Kluyveromyces thermotolerans; Nb, Nakaseomyces bacillisporus; Nd, Nakaseomyces delphensis; Sca, Saccharomyces castellii; Sc, Saccharomyces cerevisiae; Sp, Saccharomyces pastorianus; Ss, Saccharomyces servazzii; Vp, Vanderwaltozyma polyspora.

Figure 6.

Threonylation of and tRNA^His variants (3 µM) by C. albicans and S. pombe MST1 (0.3 µM). (A) CaMST1 and SpMST1 threonylate but not . (B and C) CaMST1 and SpMST1 are unable to threonylate tRNA^His variants.

Figure 7.

Proposed model for CUN codon reassignment in yeast mitochondria. (1) tRNA^His duplicated in an ancestral yeast species while CUN codons remain assigned to Leu. (2) CUN codons were changed to UUA or UUG decoded by , and was lost. (3) tRNA^His evolved to , and CUN codons reemerged from various codons.