Mistakes in translation: Reflections on mechanism

Abstract

Mistakes in translation of messenger RNA into protein are clearly a detriment to the recombinant production of pure proteins for biophysical study or the biopharmaceutical market. However, they may also provide insight into mechanistic details of the translation process. Mistakes often involve the substitution of an amino acid having an abundant codon for one having a rare codon, differing by substitution of a G base by an A base, as in the case of substitution of a lysine (AAA) for arginine (AGA). In these cases one expects the substitution frequency to depend on the relative abundances of the respective tRNAs, and thus, one might expect frequencies to be similar for all sites having the same rare codon. Here we demonstrate that, for the ADP-ribosylation factor from yeast expressed in E. coli, lysine for arginine substitutions frequencies are not the same at the 9 sites containing a rare arginine codon; mis-incorporation frequencies instead vary from less than 1 to 16%. We suggest that the context in which the codons occur (clustering of rare sites) may be responsible for the variation. The method employed to determine the frequency of mis-incorporation involves a novel mass spectrometric analysis of the products from the parallel expression of wild type and codon-optimized genes in 15N and 14N enriched media, respectively. The high sensitivity and low material requirements of the method make this a promising technology for the collection of data relevant to other mis-incorporations. The additional data could be of value in refining models for the ribosomal translation elongation process.

Fig 1. DNA sequences for wild type (WT_yARF1) and codon optimized (CO_yARF1) yeast ARF1.

Numbering is for the corresponding amino acids beginning after the initial methionine. The protein sequence of the wild type protein is provided to facilitate translation. Red denotes an arginine codon that is rare in E. coli. Blue denotes a leucine codon that is rare in E. coli. Green denotes the more E. coli abundant arginine codon substituted in CO_yARF1. It is rare in yeast, but it also exists in two sites for WT_yARF1. Yellow denotes the more E. coli abundant leucine codon substituted in CO_yARF1.

Fig 2. Mass spectrum of full-length yARF1.

The non-substituted peak is at 21464 Da. The two substituted peaks are at 21436 Da (one substitution) and 21407 Da (two substitutions).

Fig 3. Mass spectrum of WT peptide 83–96.

The peak from codon-optimized unlabeled yARF1 is labeled “Light”. The peak from unsubstituted ¹⁵N-labeled wild-type yARF1 is labeled “Heavy”, as is the peak from the R96→K substitution product. The m/z where the codon-optimized “Light” substitution product would appear (1536.75) shows no peak for any peptide measured.

Fig 4

Heavy to light ratios for (A) WT and (B) D95A yARF1. Error bars represent two standard deviations from the mean. In each case, the charge state with the lowest local chemical noise level was chosen, except for peptide 83–96 where the ¹³C isotopic envelope is distorted because of spectral overlap. Also, in the WT 83–96 peptide, the Heavy peptide overlaps with the substituted Heavy 59–72 peptide, which may lead to under-estimation of substitution.