Synonymous codon substitutions regulate transcription and translation of an upstream gene

Synonymous codons were originally viewed as interchangeable with no phenotypic consequences. However, over the years a substantial body of evidence has demonstrated that some synonymous substitutions can perturb a variety of gene expression and protein homeostasis mechanisms, including translational efficiency, translational fidelity and co-translational folding of the encoded protein. To date, synonymous codon-derived perturbations have largely focused on effects within a single gene. Here we show that synonymous codon substitutions made far within an E. coli plasmid-encoded protein coding sequence frequently led to significant upregulation of a neighboring, upstream gene. Notably, in four out of nine synonymously recoded sequences, significant upregulation of the upstream gene arose due to cryptic transcription of the anti-sense strand. Surprisingly, cryptic transcription of the upstream gene readily bypassed its native transcriptional repression mechanism. Even more surprisingly, translation of this upstream gene correlates closely with the subset of its mRNA transcribed from the cryptic internal promoter, rather than its total mRNA level. These results suggest that synonymous codons in bacteria may be under selection to both preserve the amino acid sequence of the encoded gene while also avoiding internal sequence elements that significantly perturb transcriptional and translational regulation of neighboring genes. ### Competing Interest Statement The authors have declared no competing interest.