Reduced amino acid specificity of mammalian tyrosyl-tRNA synthetase is associated with elevated mistranslation of Tyr codons.
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.
From the Department of Microbiology, Ohio State Biochemistry Program, and Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210-1292 and.
Medha
M
Raina
From the Department of Microbiology, Ohio State Biochemistry Program, and Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210-1292 and.
Adil
A
Moghal
Amgen Incorporated, Thousand Oaks, California 91320-1799.
Amanda
A
Kano
Amgen Incorporated, Thousand Oaks, California 91320-1799.
Mathew
M
Jerums
Amgen Incorporated, Thousand Oaks, California 91320-1799.
Paul D
PD
Schnier
Amgen Incorporated, Thousand Oaks, California 91320-1799.
Shun
S
Luo
Amgen Incorporated, Thousand Oaks, California 91320-1799.
Rohini
R
Deshpande
Amgen Incorporated, Thousand Oaks, California 91320-1799.
Pavel V
PV
Bondarenko
Amgen Incorporated, Thousand Oaks, California 91320-1799.
Henry
H
Lin
From the Department of Microbiology, Ohio State Biochemistry Program, and Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210-1292 and ibba.1@osu.edu.
Michael
M
Ibba
D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D003062:Codon; D006224:Cricetinae; D003412:Cricetulus; D014176:Protein Biosynthesis; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*
D003062:Codon; D014448:Tyrosine-tRNA Ligase
Aminoacyl tRNA Synthesis; Aminoacyl tRNA Synthetase; Protein Synthesis; Transfer RNA (tRNA); Translation