Getting the message: expansion of a bacterial three-hybrid assay to mRNA-protein interactions

Non‐coding small RNAs (sRNAs) contribute to bacterial biofilm formation and virulence, but there is much we do not understand about their molecular mechanisms. Hfq is the best‐studied bacterial RNA chaperone protein and has become the paradigmatic example for how RNA‐binding proteins facilitate mRNA regulation by sRNAs. Here we present progress on establishing and broadening the capabilities of a recently developed bacterial three‐hybrid (B3H) assay for studying interactions between Hfq and its RNA targets. While Hfq interactions with both mRNAs and sRNAs are critical for its function, only sRNA‐Hfq interactions have been accessible in the B3H assay to date. In order to expand the assay to mRNA‐Hfq interactions, we first demonstrated the assay’s capacity to detect interactions between synthetic mRNA mimics and Escherichia coli Hfq. Furthermore, we have developed constructs that support detection of interactions between mRNA 5′UTRs and two E. coli RNA‐chaperone proteins: Hfq and ProQ. This development points to the potential of the B3H assay to detect a wider variety of RNA‐protein interactions than previously possible, and will allow us to dissect the molecular mechanisms of these interactions. Current efforts aim to understand the relationship between the translational status of an mRNA fragment and its ability to bind protein partners in the B3H assay. In addition, we are working to understand the relationship between the in vivo B3H data on RNA‐protein interactions and the binding energetics of these interactions, which are typically measured in vitro . The results of these efforts will provide a deeper understanding of the implications of B3H data for RNA‐protein binding and regulatory mechanisms. By expanding both the capabilities of this bacterial three‐hybrid assay and our understanding of the data it provides, we aim to broaden its utility in studying bacterial gene regulation and RNA‐protein interactions. Support or Funding Information This work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.