Computational Design of Prokaryotic Translation Regulation Based on RNA-Binding Proteins

Translation regulation enables precise control of protein expression levels. In prokaryotes, regulatory RNAs and RNA-binding proteins (RBPs) can modulate ribosome activity at the level of translation initiation. Here, we examine the computational design of RBP-based translation regulation systems. We first present a systematic exploration of protein databases and genomic datasets to identify candidate regulatory RBPs. We then describe the design of target RNA sequences required to achieve distinct regulatory behaviors. We show how model-based experimental characterization provides quantitative insight into regulatory performance and enables rational comparison among different designs. Finally, we present omics-based approaches to evaluate the orthogonality of the engineered systems within the host organism. Overall, our procedure outlines the main stages for engineering RBP-mediated translation control and provides a synthetic biology framework for programming bacterial cells.