Coding-Sequence Determinants of Gene Expression in <i>Escherichia coli</i>

TLDR

Synonymous mutations do not change the protein sequence but can affect gene expression. The study aimed to determine how synonymous variation influences expression by creating a synthetic library of 154 GFP‑encoding genes with random synonymous changes. The library was expressed in *Escherichia coli* to measure GFP protein, mRNA levels, degradation patterns, and growth rates. GFP protein levels varied 250‑fold, and mRNA abundance, degradation, and growth rates also fluctuated, but codon bias showed no correlation, while mRNA folding stability near the ribosomal binding site explained over half of the protein variation, indicating that translation initiation rates dominate individual gene expression while codon bias affects overall translation efficiency and fitness.

Abstract

Synonymous mutations do not alter the encoded protein, but they can influence gene expression. To investigate how, we engineered a synthetic library of 154 genes that varied randomly at synonymous sites, but all encoded the same green fluorescent protein (GFP). When expressed in Escherichia coli, GFP protein levels varied 250-fold across the library. GFP messenger RNA (mRNA) levels, mRNA degradation patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Rather, the stability of mRNA folding near the ribosomal binding site explained more than half the variation in protein levels. In our analysis, mRNA folding and associated rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias influences global translation efficiency and cellular fitness.