Reprogramming Eukaryotic Translation with Ligand-Responsive Synthetic RNA Switches

Protein synthesis in eukaryotes is regulated by diverse reprogramming mechanisms that expand the coding capacity of individual genes. Here, we exploit one such mechanism termed −1 programmed ribosomal frameshifting (−1 PRF) to engineer ligand-responsive RNA switches that regulate protein expression. First, efficient −1 PRF stimulatory RNA elements were discovered by in vitro selection; then, ligand-responsive switches were constructed by coupling −1 PRF stimulatory elements to RNA aptamers using rational design and in vivo directed evolution. We demonstrate that −1 PRF switches tightly control the relative stoichiometry of two distinct protein outputs from a single mRNA, exhibiting consistent ligand response across whole populations of cells. Furthermore, −1 PRF switches were applied to build single-mRNA logic gates and an apoptosis module in yeast. Together, these results showcase the potential for harnessing translation-reprogramming mechanisms for synthetic biology, and establish −1 PRF switches as powerful RNA tools for controlling protein synthesis in eukaryotes.