Causes and effects of N-terminal codon bias in bacterial genes. Escherichia coli str. K-12 substr. MG1655

The genetic code allows multiple codons to encode an amino acid, and codon choice has strong effects on protein expression. Rare codons are enriched at the N-terminus of genes in most organisms, though the causes and effects of this bias are unclear. Here, we measure expression from >14,000 synthetic reporters in E. coli and show that using N-terminal rare codons instead of common ones increases expression by ~14-fold (median 4-fold). We quantify how individual N-terminal codons affect expression and show that these effects shape the sequence of natural genes. Finally, we demonstrate that reduced RNA structure and not codon rarity itself is responsible for expression increases. Our observations resolve controversies over the roles of N-terminal codon bias and suggest a straightforward method for optimizing heterologous gene expression in bacteria.