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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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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) tRNAHis with a G−1 (circled, a major anti-determinant for ThrRS). The primary sequences of  and tRNAHis are 72% identical (shaded).
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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) tRNAHis with a G−1 (circled, a major anti-determinant for ThrRS). The primary sequences of and tRNAHis are 72% identical (shaded).

Mentions: One last codon reassignment in the well-studied organism Saccharomyces cerevisiae is still not understood. In certain budding yeasts (including Saccharomyces, Nakaseomyces and Vanderwaltozyma) the four CUN (N denotes U, C, A or G) codons in the mitochondria are reassigned from leucine (Leu) to threonine (Thr) (11,12). This results from the loss of (with a UAG anticodon) that would translate CUN codons, and from the presence of an abnormal with an enlarged 8-nt anticodon loop and a UAG anticodon (Figure 1) (11,13). This codon sense change was confirmed by protein sequencing of the S. cerevisiae mitochondrial ATPase (14). Mass spectrometry studies have also validated that at least three CUU and two CUA codons in S. cerevisiae mitochondrial-encoded proteins are recoded as Thr (15). In addition to , yeast mitochondria also express a normal with a UGU anticodon that reads the ACN threonine codon box (Figure 1). It was shown previously that while mitochondrial extracts from a wild-type (WT) S. cerevisiae strain could attach Thr to both and , extracts from an MST1 mutant strain could only threonylate but not (16). Thus it was suggested that MST1 serves as a mitochondrial threonyl-tRNA synthetase (ThrRS) specific for aminoacylation of , while a different ThrRS aminoacylates in the mitochondria. Three decades after this discovery it is still a mystery how the unusual emerged in the mitochondrial genome. Previous hypotheses suggest that might have evolved from , or alternatively from the missing (11,17,18). However, both hypotheses lack convincing experimental evidence. To our surprise, biochemical and phylogenetic analyses demonstrate that directly evolved from mitochondrial tRNAHis. Saccharomyces cerevisiae mitochondrial tRNAHis shares high (72%) sequence identity with , and a single-nucleotide change converts tRNAHis to a substrate for MST1. Our work thus resolves the long-standing question regarding the origin of and its coding response.Figure 1.


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)

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) tRNAHis with a G−1 (circled, a major anti-determinant for ThrRS). The primary sequences of  and tRNAHis are 72% identical (shaded).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113583&req=5

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) tRNAHis with a G−1 (circled, a major anti-determinant for ThrRS). The primary sequences of and tRNAHis are 72% identical (shaded).
Mentions: One last codon reassignment in the well-studied organism Saccharomyces cerevisiae is still not understood. In certain budding yeasts (including Saccharomyces, Nakaseomyces and Vanderwaltozyma) the four CUN (N denotes U, C, A or G) codons in the mitochondria are reassigned from leucine (Leu) to threonine (Thr) (11,12). This results from the loss of (with a UAG anticodon) that would translate CUN codons, and from the presence of an abnormal with an enlarged 8-nt anticodon loop and a UAG anticodon (Figure 1) (11,13). This codon sense change was confirmed by protein sequencing of the S. cerevisiae mitochondrial ATPase (14). Mass spectrometry studies have also validated that at least three CUU and two CUA codons in S. cerevisiae mitochondrial-encoded proteins are recoded as Thr (15). In addition to , yeast mitochondria also express a normal with a UGU anticodon that reads the ACN threonine codon box (Figure 1). It was shown previously that while mitochondrial extracts from a wild-type (WT) S. cerevisiae strain could attach Thr to both and , extracts from an MST1 mutant strain could only threonylate but not (16). Thus it was suggested that MST1 serves as a mitochondrial threonyl-tRNA synthetase (ThrRS) specific for aminoacylation of , while a different ThrRS aminoacylates in the mitochondria. Three decades after this discovery it is still a mystery how the unusual emerged in the mitochondrial genome. Previous hypotheses suggest that might have evolved from , or alternatively from the missing (11,17,18). However, both hypotheses lack convincing experimental evidence. To our surprise, biochemical and phylogenetic analyses demonstrate that directly evolved from mitochondrial tRNAHis. Saccharomyces cerevisiae mitochondrial tRNAHis shares high (72%) sequence identity with , and a single-nucleotide change converts tRNAHis to a substrate for MST1. Our work thus resolves the long-standing question regarding the origin of and its coding response.Figure 1.

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