Engineering the 5′ UTR-Mediated Regulation of Protein Abundance in Yeast Using Nucleotide Sequence Activity Relationships

The 5' untranslated region (5'UTR) plays a key role in post-transcriptional regulation, but interaction between nucleotides and directed evolution of S'UTRs as synthetic regulatory elements remain unclear. By constructing a library of synthesized random 5'UTRs of 24 nucleotides in Saccharomyces cerevisiae, we observed strong epistatic interactions among bases from different positions in the 5'UTR. Taking into account these base interactions, we constructed a mathematical model to predict protein abundance with a precision of R-2 = 0.60. On the basis of this model, we developed an approach to engineer 5'UTRs according to nucleotide sequence activity relationships (NuSAR), in which 5'UTRs were engineered stepwise through repeated cycles of backbone design, directed screening, and model reconstruction. After three rounds of NuSAR, the predictive accuracy of our model was improved to R-2 = 0.71, and a strong 5'UTR was obtained with 5-fold higher protein abundance than the starting 5'UTR. Our findings provide new insights into the mechanism of S'UTR regulation and contribute to a new translational elements engineering approach in synthetic biology.