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.

Broad range of missense error frequencies in cellular proteins

“…For instance, the mutation R378A results in a 10-fold larger destabilization of the EF-Tu–aminoacyl-tRNA complex, compared to mutations at positions R59 and R283 (69). Similar qualitative observations of context-dependent errors were reported for other model proteins expressed under stress conditions, and the effect was attributed to differences in protein stability (19) or in the local lower accuracy of translation due to rare codon clusters (70). Thus, in the few cases for which quantitative information is available, the observed lower fidelity correlates with a higher complex stability.…”

Molecular analysis of Taro and Bali cattle using cytochrome oxidase subunit I (COI) in Indonesia

“…The cytochrome-C oxidase genes were coded for functional cytochrome amino acids (Pierron et al 2014;Skibinski et al 2017;Sabir et al 2019). The amino acid composition of the protein encoded in the COI gene rarely undergoes substitution (Quax et al 2015;Liu et al 2017). Therefore, the COI gene was stable and used as a marker for phylogeny analysis; however, the bases in the triple codon were still changing (Buhay 2009;Muyle et al 2011;Wahlberg and Wheat 2008).…”

Potential role of the X circular code in the regulation of gene expression

“…Does the self-complementary property of the X circular code contribute to coordination between X motifs in mRNA and X motifs in tRNA and/or rRNA? So far we have mainly discussed the effects of codon choices on the throughput of translation, but changes in the translation elongation process can clearly affect translation fidelity and accuracy, 13 reviewed in [63] . For example, clustering of rare codons could deplete cognate tRNAs, increasing the probability of a near-or non-cognate tRNA occupying the decoding site, and this probability could be reflected in the frequency of miss-incorporation.…”