Optimization of a bacterial-three hybrid assay for discovery and characterization of sRNA-binding proteins

Non‐coding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by protein chaperones. Hfq is an RNA chaperone protein that, through RNA interactions on distinct surfaces of its hexameric structure, supports many sRNAs to regulate translation and stability of their mRNA targets. Though Hfq is well characterized in E. coli and some additional model bacteria, Hfq homologs have only been identified in ~50% of sequenced bacteria, leading to speculation that additional RNA chaperone proteins may play important roles in sRNA function in other bacteria. 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 and protein stabilizes RNA polymerase at a test promoter and activates reporter gene expression. While this assay current detects numerous high‐affinity RNA‐protein interactions, the signal‐to‐noise makes it challenging to detect weaker interactions. In order to lay the foundation to search for new bacterial RNA‐binding proteins, we are using Hfq‐sRNA interactions as a model system to optimize the system, so that weaker RNA‐protein interactions can be reliably detected. Here we will present our efforts to improve the assay’s signal‐to‐noise ratio by adjusting the sequence, concentration, and spacing of different components within the B3H assay. With these improvements, we will work to identify novel protein‐RNA interactions, as well as dissect the binding mechanisms of these interactions. Support or Funding Information This work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.