Abstract
Quality control operates at different steps in translation to limit errors to approximately one mistranslated codon per 10,000 codons during mRNA-directed protein synthesis. Recent studies have suggested that error rates may actually vary considerably during translation under different growth conditions. Here we examined the misincorporation of Phe at Tyr codons during synthesis of a recombinant antibody produced in tyrosine-limited Chinese hamster ovary (CHO) cells. Tyr to Phe replacements were previously found to occur throughout the antibody at a rate of up to 0.7% irrespective of the identity or context of the Tyr codon translated. Despite this comparatively high mistranslation rate, no significant change in cellular viability was observed. Monitoring of Phe and Tyr levels revealed that changes in error rates correlated with changes in amino acid pools, suggesting that mischarging of tRNA(Tyr) with noncognate Phe by tyrosyl-tRNA synthetase was responsible for mistranslation. Steady-state kinetic analyses of CHO cytoplasmic tyrosyl-tRNA synthetase revealed a 25-fold lower specificity for Tyr over Phe as compared with previously characterized bacterial enzymes, consistent with the observed increase in translation error rates during tyrosine limitation. Functional comparisons of mammalian and bacterial tyrosyl-tRNA synthetase revealed key differences at residues responsible for amino acid recognition, highlighting differences in evolutionary constraints for translation quality control.
Highlights
Translation of Tyr codons is highly prone to Phe misincorporation during amino acid limitation in Chinese hamster ovary (CHO) cells
We show that CHO TyrRS has a lower than expected specificity for Tyr over Phe, consistent with the previously observed error rates [11]
Residues lost from the eukaryotic enzyme but present in the bacterial counterpart were found to be important for substrate recognition and discrimination, illustrating how different evolutionary constraints have shaped the specificities of bacterial and higher eukaryotic TyrRS
Summary
Translation of Tyr codons is highly prone to Phe misincorporation during amino acid limitation in CHO cells. Steady-state kinetic analyses of CHO cytoplasmic tyrosyl-tRNA synthetase revealed a 25-fold lower specificity for Tyr over Phe as compared with previously characterized bacterial enzymes, consistent with the observed increase in translation error rates during tyrosine limitation. By aminoacyl tRNA synthetases (aaRSs), binding of elongation factor 1A (EF 1A) to the cognate aa-tRNA, and the selection of the correct aa-tRNA by the ribosome All these steps have their own inherent error rate, which is thought to vary depending on various environmental conditions. Quality control at different steps can limit errors to approximately one mistranslated codon per 10,000 during mRNA-directed protein synthesis [1, 2, 8], recent studies suggest that error rates vary considerably during translation. Residues lost from the eukaryotic enzyme but present in the bacterial counterpart were found to be important for substrate recognition and discrimination, illustrating how different evolutionary constraints have shaped the specificities of bacterial and higher eukaryotic TyrRS
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