Good Codons, Bad Transcript: Large Reductions in Gene Expression and Fitness Arising from Synonymous Mutations in a Key Enzyme

Abstract

Biased codon usage in protein-coding genes is pervasive, whereby amino acids are largely encoded by a specific subset of possible codons. Within individual genes, codon bias is stronger at evolutionarily conserved residues, favoring codons recognized by abundant tRNAs. Although this observation suggests an overall pattern of selection for translation speed and/or accuracy, other work indicates that transcript structure or binding motifs drive codon usage. However, our understanding of codon bias evolution is constrained by limited experimental data on the fitness effects of altering codons in functional genes. To bridge this gap, we generated synonymous variants of a key enzyme-coding gene in Methylobacterium extorquens. We found that mutant gene expression, enzyme production, enzyme activity, and fitness were all significantly lower than wild-type. Surprisingly, encoding the gene using only rare codons decreased fitness by 40%, whereas an allele coded entirely by frequent codons decreased fitness by more than 90%. Increasing gene expression restored mutant fitness to varying degrees, demonstrating that the fitness disadvantage of synonymous mutants arose from a lack of beneficial protein rather than costs of protein production. Protein production was negatively correlated with the frequency of motifs with high affinity for the anti-Shine-Dalgarno sequence, suggesting ribosome pausing as the dominant cause of low mutant fitness. Together, our data support the idea that, although a particular set of codons are favored on average across a genome, in an individual gene selection can either act for or against codons depending on their local context.

Adaptive synonymous mutations in an experimentally evolved Pseudomonas fluorescens population

“…fitness advantage relative to the ancestor (Fig. 1c), values that are substantially larger than any previous estimate of fitness for a synonymous mutation 3,[8][9][10][11] .…”

“…Consistent with this expectation, estimates of fitness from comparative genomics suggest that synonymous sites are under weak purifying selection [13][14][15] , although a recent study suggests that up to 22% of synonymous sites in the Drosophila melanogaster genome may be under strong purifying selection 16 . Direct measures of fitness from site-directed mutagenesis studies tell a similar story: the fitness effects of synonymous mutations are almost always deleterious or, more rarely, weakly beneficial 3,[8][9][10][11] . Deleterious mutations are, by definition, subject to purifying selection, while weakly beneficial mutations are unlikely to be substituted by selection because either they cannot escape drift in small populations or, in large populations, are eliminated in competition with the relatively more abundant class of non-synonymous mutations with larger beneficial effects.…”

“…Pervasive variation in the frequencies of synonymous codons across genes and genomes (codon usage bias) 1 suggests that selection may favour some synonymous codons over others. Moreover, direct measures of the impact of synonymous mutations from nucleotide replacement studies demonstrate that they can impact gene expression 2,3 , presumably through changes to the rate or accuracy of transcription and/or translation 4 , mRNA stability and folding 5 , protein secondary structure 6,7 and even fitness 3,[8][9][10][11] . Taken together, these results suggest that the frequency of at least some synonymous mutations is governed by selection.…”

Recurrent positive selection and heterogeneous codon usage bias events leading to coexistence of divergent pigeon circoviruses

“…(Shabalina et al, 2013). Bacterial experiments have shown that the use of single or rare codons could impose fitness disadvantage than using various codons for translation (Agashe et al, 2013), which explains the divergent codon usages of capsid genes in Taiwanese PiCVs.…”

Experimental evolution of Escherichia coli harboring an ancient translation protein

“…(Color figure online) researchers to address the role of chance and necessity at the molecular level? The key to resolving these prior questions is, at least in part, to assess the degree to which our system tracks or differs from experimental systems that replace genomic components with homologs obtained from other extant organisms (Acevedo-Rocha et al 2013;Agashe et al 2013;Andersson and Hughes 2009;Pena et al 2010;Urbanczyk et al 2012).…”