Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 289, Issue 25, Pages 17780-17790Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M114.564609
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Funding
- National Science Foundation (NSF) [MCB-1052344]
- NSF-Grant Opportunities for Academic Liaison with Industry (NSF-GOALI) fellowship
- NIH [GM008512, GM086252]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1412611] Funding Source: National Science Foundation
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1052344] Funding Source: National Science Foundation
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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.
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