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Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid.

Kwon I, Choi ES - PLoS ONE (2016)

Bottom Line: However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons.Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites.Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.

View Article: PubMed Central - PubMed

Affiliation: School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea.

ABSTRACT
Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.

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Extended wobble rules (A). Mutant ytRNAPheAAA recognizing UUU and UUC Phe codons by Watson-Crick (W/C) base pairing and wobble base pairing, respectively (B). Mutant ytRNAPheCAA recognizing UUG Leu codon by W/C base pairing but none of other Leu codons (C).
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pone.0152826.g002: Extended wobble rules (A). Mutant ytRNAPheAAA recognizing UUU and UUC Phe codons by Watson-Crick (W/C) base pairing and wobble base pairing, respectively (B). Mutant ytRNAPheCAA recognizing UUG Leu codon by W/C base pairing but none of other Leu codons (C).

Mentions: We reasoned that misincorporation of 2Nal at the UUC codon resulted from the recognition of UUC codons by the AAA anticodon of ytRNAPheAAA. According to Crick’s wobble rule proposed in 1966 [29], the base A in the first position of the anticodon can recognize only the base U in the third position of the codon. Therefore, the UUC codon should not be recognized by the AAA anticodon. The discrepancy between the experimental results and Crick’s wobble rule may be explained by the expanded wobble rule proposed by Lim and Curran in 2001 [30]. The expanded wobble rule is based on new experimental findings [30–37] and stereochemical modeling [38–41] of codon-anticodon interactions. According to the expanded wobble rule (Fig 2A), A in the first position of the anticodon can recognize all four bases in the third position in the codon. The base A in the first position of the anticodon favors bases in the order U > C > G > A (Fig 2B), consistent with the codon-biased incorporation of 2Nal observed in this work.


Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid.

Kwon I, Choi ES - PLoS ONE (2016)

Extended wobble rules (A). Mutant ytRNAPheAAA recognizing UUU and UUC Phe codons by Watson-Crick (W/C) base pairing and wobble base pairing, respectively (B). Mutant ytRNAPheCAA recognizing UUG Leu codon by W/C base pairing but none of other Leu codons (C).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814082&req=5

pone.0152826.g002: Extended wobble rules (A). Mutant ytRNAPheAAA recognizing UUU and UUC Phe codons by Watson-Crick (W/C) base pairing and wobble base pairing, respectively (B). Mutant ytRNAPheCAA recognizing UUG Leu codon by W/C base pairing but none of other Leu codons (C).
Mentions: We reasoned that misincorporation of 2Nal at the UUC codon resulted from the recognition of UUC codons by the AAA anticodon of ytRNAPheAAA. According to Crick’s wobble rule proposed in 1966 [29], the base A in the first position of the anticodon can recognize only the base U in the third position of the codon. Therefore, the UUC codon should not be recognized by the AAA anticodon. The discrepancy between the experimental results and Crick’s wobble rule may be explained by the expanded wobble rule proposed by Lim and Curran in 2001 [30]. The expanded wobble rule is based on new experimental findings [30–37] and stereochemical modeling [38–41] of codon-anticodon interactions. According to the expanded wobble rule (Fig 2A), A in the first position of the anticodon can recognize all four bases in the third position in the codon. The base A in the first position of the anticodon favors bases in the order U > C > G > A (Fig 2B), consistent with the codon-biased incorporation of 2Nal observed in this work.

Bottom Line: However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons.Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites.Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.

View Article: PubMed Central - PubMed

Affiliation: School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea.

ABSTRACT
Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.

Show MeSH
Related in: MedlinePlus