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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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Threonylation of  and tRNAHis 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 tRNAHis variants.
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Figure 6: Threonylation of and tRNAHis 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 tRNAHis variants.

Mentions: The biochemical and phylogenetic evidence above suggests that has evolved from mitochondrial tRNAHis, at a time point close to the divergence of Kluyveromyces species. To understand whether could be recognized by MST1 enzymes from other fungal species, we overexpressed and purified MST1s from C. albicans and S. pombe [a non-hemiascomycete ‘fission yeast’ belonging to Taphrinomycotina (37)], and tested them for threonylation of . CaMST1 and SpMST1 share 49 and 43% sequence identity with ScMST1, respectively, and both enzymes were able to aminoacylate S. cerevisiae mitochondrial (Figure 6A). However, neither CaMST1 nor SpMST1 recognized or the tRNAHis variants tested above (Figure 6). These results strongly suggest that MST1 specifically evolved to recognize in a group of yeasts comprising S. cerevisiae. Therefore, CUN codon reassignment was completed following the coevolution of MST1 and , which established specific protein–tRNA interactions.Figure 6.


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)

Threonylation of  and tRNAHis 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 tRNAHis variants.
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Related In: Results  -  Collection

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Figure 6: Threonylation of and tRNAHis 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 tRNAHis variants.
Mentions: The biochemical and phylogenetic evidence above suggests that has evolved from mitochondrial tRNAHis, at a time point close to the divergence of Kluyveromyces species. To understand whether could be recognized by MST1 enzymes from other fungal species, we overexpressed and purified MST1s from C. albicans and S. pombe [a non-hemiascomycete ‘fission yeast’ belonging to Taphrinomycotina (37)], and tested them for threonylation of . CaMST1 and SpMST1 share 49 and 43% sequence identity with ScMST1, respectively, and both enzymes were able to aminoacylate S. cerevisiae mitochondrial (Figure 6A). However, neither CaMST1 nor SpMST1 recognized or the tRNAHis variants tested above (Figure 6). These results strongly suggest that MST1 specifically evolved to recognize in a group of yeasts comprising S. cerevisiae. Therefore, CUN codon reassignment was completed following the coevolution of MST1 and , which established specific protein–tRNA interactions.Figure 6.

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.

Show MeSH