The Effects of Silent Mutations on Protein Expression and Stability

The genetic code is redundant, where several alternative codons code for a single amino acid. Synonymous codons are translated at different rates by the ribosome, with some codons considered to be “fast-translating” and others to be “slow- translating”. Since many proteins fold co-translationally, synonymous codon usage, and the resulting translation speed, was suggested to influence nascent chain folding. A proposed underlying mechanism suggests that pausing between the translation of independently-folding units supports their correct folding. The importance of codon usage to protein folding and function was shown for a few individual cases, but a clear and general understanding of the phenomenon is still missing. In this work, we aim to identify conserved regions of slow codons that are important for correct protein folding and function.

To this end, we analyzed 1,115 orthologue protein groups from E. coli and B. subtilis to identify slow codon stretches that appear to be important for a specific 3-D protein fold. Based on this analysis, we selected several proteins for experimental validation. We generated a modified version of each gene in which the slow codon stretch was replaced with a synonymous fast one. We compared the modified version to the wild type one in a set of experiments checking expression, solubility and function. Preliminary results show that in several cases, especially in oligomeric proteins, strategically positioned slow codons are important for protein stability and function, suggesting that biased codon usage is a general mechanism that assists correct protein folding.