Boosting Gene Translation by a Short ORF Encoding for a “Nonsense” Peptide Positioned Immediately Upstream

We demonstrate a new synthetic genetic component for boosting translational levels in bacteria. Boosting is facilitated by a short and nonfunctional open reading frame (nfORF) that encodes a "nonsense" peptide located upstream of the target gene. The nfORF may be either in-frame or frame-shifted relative to the target open reading frame (tORF), and it contains an internal strong RBS that is immediately adjacent to its stop codon. We characterized 50 nfORFs, which allowed us to build a predictive model for the nfORF sequence, length, and distance from the tORF. Finally, we validated the model by testing three additional unseen nfORFs and demonstrated that the model also accurately predicts a translational boost for another target gene. Our results show how ribosome recruitment can be engineered to boost translation by as much as ×20 by precise engineering of an upstream nfORF. Our finding can be used for a variety of gene expression applications, by increasing expression for potentially any target gene that is produced in <i>Escherichia coli</i>.