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 Escherichia coli.