Optimization of a Bacterial Three‐Hybrid Assay for RNA‐Protein Interactions

Abstract

Non‐coding RNAs regulate gene expression in every domain of life. In bacteria, non‐coding small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by protein chaperones. While much is known about the mechanism of a few model RNA chaperone proteins, significant gaps remain in our understanding of how other bacterial RNA‐binding proteins interact with RNA to regulate gene expression. While there are numerous biochemical methods to study bacterial RNA‐protein interactions, new genetic methodologies would be a powerful complement to current tools. We have recently developed a bacterial three‐hybrid (B3H) assay that detects the binding of E. coli sRNAs with multiple RNA chaperone proteins. This assay couples the transcription of a genetic reporter to the interaction of a DNA‐bound “bait” RNA (e.g. an sRNA) and an RNAP‐fused “prey” protein (e.g. Hfq). Successful interaction between RNA bait and protein prey stabilizes RNA polymerase (RNAP) at a test promoter and activates reporter gene expression. This B3H assay offers a straightforward path to identify and interrogate mutations in these RNA‐binding proteins with molecular phenotypes of interest. Currently, only ~40% of known sRNA‐chaperone interactions are detectable in the system. In order to maximize the utility of our bacterial three‐hybrid assay, a larger proportion of known RNA‐protein interactions must be successfully detectable. Current efforts are focused on optimizing the signal of the B3H assay so that weaker RNA‐protein interactions can be reliably detected. Here, we show results from our efforts to improve the assay's signal‐to‐noise ratio by tuning the sequences, spacing, and the concentrations of the B3H assay components. We have observed an improvement in the assay's signal in response to systematic alterations of the assay components. With an optimized B3H assay, we aim to discover new sRNA‐binding proteins and uncover the regulatory roles sRNAs and these proteins play in biology.Support or Funding InformationMount Holyoke College and the Luce FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.