Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

Abstract

The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 "recalcitrant" AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional "safe replacement zone" (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.codon choice | genome editing | recoded genomes

Stumbling across the Same Phage: Comparative Genomics of Widespread Temperate Phages Infecting the Fish Pathogen Vibrio anguillarum

“…Rare codons are generally responsible for encoding transcriptional and translational properties that are distinct from those encoded by the prevalent arginine codons and therefore affecting the expression of regulatory genes [86]. In accordance with this, all four arginine amino acids which are encoded by repressor gene in the H20-like bacteriophages are translated by the rare codon AGA.…”

Synonymous Codons: Choose Wisely for Expression

“…Although most substitutions were successful, most also resulted in reduced fitness. In a parallel approach, the 123 rare Arg codons (AGG and AGA) were replaced in all essential genes in E. coli [122]. Replacement of 110 rare Arg codons was straightforward, whereas the remaining 13 required alternative strategies which implicated mRNA secondary structure near the N-terminus and ribosome-binding sequences as determinants of codon choice.…”

“…Replacement of 110 rare Arg codons was straightforward, whereas the remaining 13 required alternative strategies which implicated mRNA secondary structure near the N-terminus and ribosome-binding sequences as determinants of codon choice. However, other parameters may constrain the choice of rare Arg codons at particular locations, since the resulting strain also exhibited slow growth and carried over 400 spontaneous non-synonymous mutations [122]. Thus, suboptimal codons can have important and substantive effects, but most of these remain to be discovered.…”

Rewriting the Genetic Code

“…Since the launch of this strain, several derivative strains have been developed for optimized assignments of each ncAA species (Supplemental Table 2). In separate work, coselection-MAGE (CoS-MAGE) was developed for enhancing scarless genome modification (69, 147) and was employed to eliminate all AGA/AGG arginine (Arg) rare codons (123 total) in the essential genes in E. coli (102) (Supplemental Table 2). …”