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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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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 tRNAHis variants (3 µM) by ScMST1 (0.3 µM). (C) Histidylation of  (3 µM) and tRNAHis variants (3 µM) by ScmtHisRS (0.3 µM).
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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 tRNAHis variants (3 µM) by ScMST1 (0.3 µM). (C) Histidylation of (3 µM) and tRNAHis variants (3 µM) by ScmtHisRS (0.3 µM).

Mentions: The mitochondria of all yeast species containing have lost the gene, leaving the only tRNA capable of reading CUN codons. Whereas S. cerevisiae mitochondrial LeuRS (ScmtLeuRS) efficiently attaches Leu to (29,30), it could not recognize (Figure 4A), confirming that the S. cerevisiae mitochondrial CUN codons are assigned to Thr instead of Leu. It is known that the WT S. cerevisiae strain attaches Thr to , and an MST1 deletion strain fails to threonylate , resulting in a respiration-deficient phenotype (16). This suggests that MST1 is a putative mitochondrial ThrRS. Compared with bacterial ThrRSs, yeast MST1 enzymes lack an N-terminal editing domain, but share homologous catalytic and tRNA anticodon binding domains. To test aminoacylation of directly, the S. cerevisiae MST1 gene was cloned into pET28a for protein overexpression in E. coli. N-terminal His-tagged MST1 was purified to homogeneity and tested in aminoacylation reactions in the presence of [3H] Thr and in vitro transcribed S. cerevisiae tRNAs. Consistent with previous in vivo results (16), ScMST1 was able to charge with Thr in vitro (Figure 4B). MST1 could also threonylate , which was unexpected as it was previously thought that a second mitochondrial ThrRS was responsible for aminoacylation. Steady-state kinetic experiments revealed that ScMST1 recognized and with high affinity, with Km values of 0.29 and 0.44 µM, respectively (Table 1), suggesting that tRNA modifications (17) are not critical for MST1 recognition. These data, together with previous in vivo results, establishes unequivocally that is indeed threonylated by MST1. While we favor that MST1 threonylates in vivo, we do not exclude the possibility that a second ThrRS activity is present in yeast mitochondria.Figure 4.


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)

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 tRNAHis variants (3 µM) by ScMST1 (0.3 µM). (C) Histidylation of  (3 µM) and tRNAHis variants (3 µM) by ScmtHisRS (0.3 µM).
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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 tRNAHis variants (3 µM) by ScMST1 (0.3 µM). (C) Histidylation of (3 µM) and tRNAHis variants (3 µM) by ScmtHisRS (0.3 µM).
Mentions: The mitochondria of all yeast species containing have lost the gene, leaving the only tRNA capable of reading CUN codons. Whereas S. cerevisiae mitochondrial LeuRS (ScmtLeuRS) efficiently attaches Leu to (29,30), it could not recognize (Figure 4A), confirming that the S. cerevisiae mitochondrial CUN codons are assigned to Thr instead of Leu. It is known that the WT S. cerevisiae strain attaches Thr to , and an MST1 deletion strain fails to threonylate , resulting in a respiration-deficient phenotype (16). This suggests that MST1 is a putative mitochondrial ThrRS. Compared with bacterial ThrRSs, yeast MST1 enzymes lack an N-terminal editing domain, but share homologous catalytic and tRNA anticodon binding domains. To test aminoacylation of directly, the S. cerevisiae MST1 gene was cloned into pET28a for protein overexpression in E. coli. N-terminal His-tagged MST1 was purified to homogeneity and tested in aminoacylation reactions in the presence of [3H] Thr and in vitro transcribed S. cerevisiae tRNAs. Consistent with previous in vivo results (16), ScMST1 was able to charge with Thr in vitro (Figure 4B). MST1 could also threonylate , which was unexpected as it was previously thought that a second mitochondrial ThrRS was responsible for aminoacylation. Steady-state kinetic experiments revealed that ScMST1 recognized and with high affinity, with Km values of 0.29 and 0.44 µM, respectively (Table 1), suggesting that tRNA modifications (17) are not critical for MST1 recognition. These data, together with previous in vivo results, establishes unequivocally that is indeed threonylated by MST1. While we favor that MST1 threonylates in vivo, we do not exclude the possibility that a second ThrRS activity is present in yeast mitochondria.Figure 4.

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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