Fitness costs of synonymous mutations in the rpsT gene can be compensated by restoring mRNA base pairing

Abstract

We previously reported that the distribution of fitness effects for non-synonymous and synonymous mutations in Salmonella typhimurium ribosomal proteins S20 and L1 are similar, suggesting that fitness constraints are present at the level of mRNA. Here we explore the hypothesis that synonymous mutations confer their fitness-reducing effect by alterating the secondary structure of the mRNA. To this end, we constructed a set of synonymous substitutions in the rpsT gene, encoding ribosomal protein S20, that are located in predicted paired regions in the mRNA and measured their effect on bacterial fitness. Our results show that for 3/9 cases tested, the reduced fitness conferred by a synonymous mutation could be fully or partly restored by introducing a second synonymous substitution that restore base pairing in a mRNA stem. In addition, random mutations in predicted paired regions had larger fitness effects than those in unpaired regions. Finally, we did not observe any correlation between fitness effects of the synonymous mutations and their rarity. These results suggest that for ribosomal protein S20, the deleterious effects of synonymous mutations are not generally due to codon usage effects, but that mRNA secondary structure is a major fitness constraint.

Figure 1. Predicted mRNA structure of part of rpsT.

Numbering begins at TTG start codon and positions with where synonymous mutations have been introduced are shown in larger font.

Figure 2. Fitness effects of synonymous mutations in rpsT.

Selection coefficients were measured in competition with isogenic wild type strains; a selection coefficient of 0 is neutral and negative values are deleterious. Error bars represent SEM.

Figure 3. Changes in codon usage frequencies were not correlated with fitness costs of mutations.

The frequencies of codons in S. typhimurium ribosomal protein genes were used to determine the ratio between the mutated and native codons. The relative codon frequency is the natural logarithm of this ratio.