Protein-RNA Binding in the 5’ UTR Up-regulate Translation

We explored the regulatory potential of RBP-RNA interactions in bacterial 5’ UTRs, via the synthetic biology approach combined with the SHAPE-Seq method. Using a library of RNA variants based on the binding sites of the coat RBPs of bacteriophages GA, MS2, PP7, and Qβ, we found a complex set of regulatory responses, including translational repression, translational stimulation, and cooperative behavior. SHAPE-Seq data for a representative construct exhibiting up-regulation, indicates a partially-folded hairpin and non-reactive upstream and downstream flanking region, which we attribute to intermediate structures that apparently block translation. RBP binding stabilizes the fully-folded hairpin state facilitating translation, suggesting that the up-regulating constructs function as protein-sensing regulatory elements. This finding is further supported by lengthening the binding-site stem, which in turn destabilizes the translationally-inactive state, and abolishes the up-regulating behavior. Finally, we find that the combination of two binding sites, positioned in the 5’ UTR and the ribosomal initiation region within the CDS of the same transcript, can yield a cooperative regulatory response. Together, we show that the interaction of an RBP with its RNA target facilitates structural changes in the RNA, which is reflected by a controllable range of binding affinities and dose response behaviors. Thus, demonstrating that RNA-RBP interactions can provide a platform for constructing gene regulatory networks that are based on translational, rather than transcriptional, regulation.