Effects of single synonymous substitutions on fold efficiency demonstrate the influence of rare codons and protein structure

Summary Codon sequences can influence proteins to misfold during cotranslational folding. Here, we develop an in vivo assay in E. coli to comprehensively study the impact of single synonymous substitutions on protein folding efficiency and apply it to the N-terminal domain of ddlA. By mapping the influence of codons along the sequence, we show that codon identity can substantially influence folding efficiency in a manner depending on structure and topology. We found that a cluster of codons in the N-terminal domain strongly impacts ddlA folding. Further analysis revealed that substitutions to rarer codons generally lead to increased folding efficiency. Consistent with this, an mRNA composed exclusively of rare codons yields higher expression and folding efficiency than one containing only commons codons. Our results highlight the importance of rare codons in cotranslational folding and the relationship between codon sequence and protein structure.