The study investigates the mechanisms underlying the influence of synonymous mutations on gene expression in *Escherichia coli*. The researchers engineered a synthetic library of 154 genes that vary randomly at synonymous sites but all encode the same green fluorescent protein (GFP). They found that GFP protein levels varied 250-fold across the library, with mRNA levels, mRNA degradation patterns, and bacterial growth rates also varying. However, codon bias did not correlate with gene expression. Instead, the stability of mRNA folding near the ribosomal binding site explained over half of the variation in protein levels. The analysis suggests that mRNA folding and associated rates of translation initiation play a predominant role in shaping gene expression levels, while codon bias influences global translation efficiency and cellular fitness. The findings challenge the traditional view that codon adaptation directly correlates with gene expression levels, proposing instead that strong expression induces selection for high codon adaptation.The study investigates the mechanisms underlying the influence of synonymous mutations on gene expression in *Escherichia coli*. The researchers engineered a synthetic library of 154 genes that vary randomly at synonymous sites but all encode the same green fluorescent protein (GFP). They found that GFP protein levels varied 250-fold across the library, with mRNA levels, mRNA degradation patterns, and bacterial growth rates also varying. However, codon bias did not correlate with gene expression. Instead, the stability of mRNA folding near the ribosomal binding site explained over half of the variation in protein levels. The analysis suggests that mRNA folding and associated rates of translation initiation play a predominant role in shaping gene expression levels, while codon bias influences global translation efficiency and cellular fitness. The findings challenge the traditional view that codon adaptation directly correlates with gene expression levels, proposing instead that strong expression induces selection for high codon adaptation.