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Synonymous codon substitutions perturb cotranslational protein folding in vivo and impair cell fitness

Significance Many proteins that are incapable of refolding in vitro 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 in vivo, leading to changes in cellular fitness. This work demonstrates that synonymous codon selection can play an important role in supporting efficient protein production in vivo. In the cell, proteins are synthesized from N to C terminus and begin to fold during translation. Cotranslational 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 in vivo. 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 in vivo. These results support a model in which synonymous codon substitutions can impair cell fitness by significantly perturbing cotranslational protein folding mechanisms, despite the chaperoning provided by the cellular protein homeostasis network.