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

Su D, Lieberman A, Lang BF, Simonovic M, Söll D, Ling J - Nucleic Acids Res. (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.

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.

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Sequence alignment of mitochondrial  and tRNAHis. Three major differences between  and tRNAHis 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.
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Figure 5: Sequence alignment of mitochondrial and tRNAHis. Three major differences between and tRNAHis 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.

Mentions: To provide experimental evidence for the evolution of , we examined recognition of tRNAHis variants by MST1. Except in a few α-proteobacteria [(31), and references therein], all known tRNAHis species contain a G at position −1, which is a critical identity element for histidyl-tRNA synthetase (HisRS) [(32,33) and references therein]. Sequence alignments of and mitochondrial tRNAHis genes revealed that G−1 addition in tRNAHis comprises one major difference between the two tRNA species (Figure 5). In vitro, ScMST1 failed to charge the WT SctRNAHis transcript with Thr, but deleting G−1 stimulated threonylation of tRNAHis by ScMST1 (Figure 4B). Steady-state kinetic data showed that ΔG−1 tRNAHis gained 4% threonylation activity of the WT (Table 1). Compared with , ΔG−1 tRNAHis displayed 10-fold lower kcat and 3-fold higher Km values for threonylation by ScMST1. In addition to G−1, other major differences between tRNAHis and include an A insertion in at position 35, and the discriminator base at position 73 (Figure 5). Changing C73 to A did not improve the threonylation efficiency of ΔG−1 tRNAHis, but inserting A35 in the anticodon loop of ΔG-1 tRNAHis further increased its threonylation activity 5-fold (Table 1). In the presence of G−1, A35 insertion did not allow threonylation of tRNAHis. These results suggest that G−1 is a major anti-determinant in tRNAHis for MST1. In line with this notion, addition of G−1 to reduced its threonylation activity 150-fold (Table 1).Figure 5.


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

Su D, Lieberman A, Lang BF, Simonovic M, Söll D, Ling J - Nucleic Acids Res. (2011)

Sequence alignment of mitochondrial  and tRNAHis. Three major differences between  and tRNAHis 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.
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Figure 5: Sequence alignment of mitochondrial and tRNAHis. Three major differences between and tRNAHis 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.
Mentions: To provide experimental evidence for the evolution of , we examined recognition of tRNAHis variants by MST1. Except in a few α-proteobacteria [(31), and references therein], all known tRNAHis species contain a G at position −1, which is a critical identity element for histidyl-tRNA synthetase (HisRS) [(32,33) and references therein]. Sequence alignments of and mitochondrial tRNAHis genes revealed that G−1 addition in tRNAHis comprises one major difference between the two tRNA species (Figure 5). In vitro, ScMST1 failed to charge the WT SctRNAHis transcript with Thr, but deleting G−1 stimulated threonylation of tRNAHis by ScMST1 (Figure 4B). Steady-state kinetic data showed that ΔG−1 tRNAHis gained 4% threonylation activity of the WT (Table 1). Compared with , ΔG−1 tRNAHis displayed 10-fold lower kcat and 3-fold higher Km values for threonylation by ScMST1. In addition to G−1, other major differences between tRNAHis and include an A insertion in at position 35, and the discriminator base at position 73 (Figure 5). Changing C73 to A did not improve the threonylation efficiency of ΔG−1 tRNAHis, but inserting A35 in the anticodon loop of ΔG-1 tRNAHis further increased its threonylation activity 5-fold (Table 1). In the presence of G−1, A35 insertion did not allow threonylation of tRNAHis. These results suggest that G−1 is a major anti-determinant in tRNAHis for MST1. In line with this notion, addition of G−1 to reduced its threonylation activity 150-fold (Table 1).Figure 5.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.

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.

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