Due to the redundancy of the genetic code most amino acids are encoded by several ‘synonymous‘ codons. These codons are used unevenly, and each organism demonstrates its own unique codon usage bias, where the ‘preferred’ codons are associated with tRNAs that are found in high concentrations. Therefore, for decades, the prevailing view had been that preferred and non-preferred codons are linked to high or slow translation rates, respectively.

However, this simplified view is contrasted by the frequent failures of codon-optimization efforts and by evidence of non-preferred (i.e. ‘slow’) codons having specific roles important for efficient production of functional proteins. One such specific role of slower codons is the regulation of co-translational protein folding, a complex biophysical process that is very challenging to model or to measure.

Here, we combined a genome-wide approach with experiments to investigate the role of slow codons in protein production and co-translational folding. We analysed homologous gene groups from divergent bacteria and identified positions of inter-species conservation of bias towards slow codons. We then generated mutants where the conserved slow codons are substituted with ‘fast’ ones, and experimentally studied the effects of these codon substitutions. Using cellular and biochemical approaches we find that at certain locations, slow-to-fast codon substitutions reduce protein expression, increase protein aggregation, and impair protein function.

This report provides an approach for identifying functionally relevant regions with slower codons and demonstrates that such codons are important for protein expression and function.