Synthetic 5’ UTRs can either up- or down-regulate expression upon RBP binding

The construction of complex gene regulatory networks requires both inhibitory and up-regulatory modules. However, the vast majority of RNA-based regulatory “parts” are inhibitory. Using a synthetic biology approach combined with SHAPE-Seq, we explored the regulatory effect of RBP-RNA interactions in bacterial 5’-UTRs. By positioning a library of RNA hairpins upstream of a reporter gene and co-expressing them with the matching RBP, we observed a set of regulatory responses, including translational stimulation, translational repression, and cooperative behavior. Our combined approach revealed three distinct states in-vivo: in the absence of RBPs, the RNA molecules can be found either in a molten state that is amenable to translation, or a structured phase that inhibits translation. In the presence of RBPs, the RNA molecules are in a semi-structured phase with partial translational capacity. Our work provides new insight into RBP-based regulation and a blueprint for designing complete gene regulatory circuits at the post-transcriptional level. Constructs containing a single hairpin, based on the binding sites of the coat RBPs of bacteriophages PP7 where tested using in-vivo (in-cell) and in-vitro SHAPE-seq experiment in duplicactes (2 replicates for in-vivo and in-vitro for each PP7-wt and PP7-USs modified or non-modified by the SHAPE reagent (NAI)= 16 samples, total)