Induced Protein-RNA Interactions in the 5’ UTR Lead to Conformational Changes that Alter Translation

Abstract

We study translational regulation by a 5’ UTR sequence encoding the binding site of an RNA-binding protein (RBP) in bacteria, using a reporter assay and selective 2′-hydroxyl acylation analysed by primer extension sequencing (SHAPE-Seq). We tested constructs containing a single hairpin, based on the binding sites of the coat RBPs of bacteriophages GA, MS2, PP7, and Qβ, positioned in the 5’ UTR of a reporter gene. With specifically-bound RBP present, either weak repression or up-regulation is observed, depending on the binding site and its flanking sequence. 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.