In vivo translational inaccuracy in Escherichia coli: Missense reporting using extremely low activity mutants of Vibrio harveyi luciferase

A convenient, sensitive assay for measurement of in vivo missense translational errors is reported that uses luciferase activity generated by mistranslation of a gene encoding an inactive mutant α chain of the Vibrio harveyi enzyme. Mutations were introduced at α45 His, a position known to be highly intolerant of amino acids other than histidine. To normalize for any variations in expression level, the concentration of wild-type luciferase αβ dimer was determined by a novel assay using co-refolding of active/wild-type β enzyme subunits with inactive α subunits in lysate with an excess of exogenously added active α subunits. Four His α45 missense mutants of luciferase encoded by leucine codons (CUC, CUU, CUG, and UUG) had histidine misincorporation rates of 2.0 × 10^-6, 1.3 × 10^-6, 9.0 × 10^-8, and 1.5 × 10 ^-8 respectively, a variation of over 133-fold among synonymous codons. Any substantial contribution of mutation was ruled out by a Luria-Delbrück fluctuation test. The two leucine codons with the highest rates, CUU and CUC, have a single central-mismatch to the histidyl-tRNA ^QUG anticodon. Aminoglycoside antibiotics known to enhance mistranslation increased the error rate of the CUC codon more than those of the CUU and CUG codons, consistent with the hypothesis that CUC codon mistranslation arises primarily from miscoding events such as the selection of noncognate histidyl-tRNA^QUG at the central position of the codon.