Synonymous codon substitutions perturb co-translational protein foldingin vivoand impair cell fitness

Abstract

<jats:title>Abstract</jats:title><jats:p>In the cell, proteins are synthesized from N- to C-terminus and begin to fold during translation. Co-translational folding mechanisms are therefore linked to elongation rate, which varies as a function of synonymous codon usage. However, synonymous codon substitutions can affect many distinct cellular processes, which has complicated attempts to deconvolve the extent to which synonymous codon usage can promote or frustrate proper protein folding <jats:italic>in vivo</jats:italic>. Although previous studies have shown that some synonymous changes can lead to different final structures, other substitutions will likely be more subtle, perturbing predominantly the protein folding pathway without radically altering the final structure. Here we show that synonymous codon substitutions encoding a single essential enzyme lead to dramatically slower cell growth. These mutations do not prevent active enzyme formation; instead, they predominantly alter the protein folding mechanism, leading to enhanced degradation <jats:italic>in vivo</jats:italic>. These results support a model where synonymous codon substitutions can impair cell fitness by significantly perturbing co-translational protein folding mechanisms, despite the chaperoning provided by the cellular protein homeostasis network.</jats:p><jats:sec><jats:title>Significance</jats:title><jats:p>Many proteins that are incapable of refolding <jats:italic>in vitro</jats:italic> nevertheless fold efficiently to their native state in the cell. This suggests that more information than the amino acid sequence is required to properly fold these proteins. Here we show that synonymous mRNA mutations can alter a protein folding mechanism <jats:italic>in vivo</jats:italic>, leading to changes in cellular fitness. This work demonstrates that synonymous codon selection can play an important role in supporting efficient protein production <jats:italic>in vivo</jats:italic>.</jats:p></jats:sec>

Genomic analysis of variability in Delta-toxin levels between Staphylococcus aureus strains

“…Strikingly, the variant discovered through the GWAS screen, and all mutations in the gene in high production CCs such as CC45 were synonymous. There is a growing body of literature documenting differences in protein function caused by synonymous mutations that impact RNA toxicity (Mittal et al, 2018) or protein folding (Walsh, Bowman & Clark, 2019). Given the involvement of the Agr system, there is possibly a role for mutations to change RNAIII binding specificity and influence gene regulation.…”