Abstract
We have previously developed a transcription-based bacterial three-hybrid (B3H) assay as a genetic approach to probe RNA-protein interactions inside of E. coli cells. This system offers a straightforward path to identify and assess the consequences of mutations in RBPs with molecular phenotypes of interest. One limiting factor in detecting RNA-protein interactions in the B3H assay is RNA misfolding arising from incorrect base-pair interactions with neighboring RNA sequences in a hybrid RNA. To support correct folding of hybrid bait RNAs, we have explored the use of a highly stable stem ("GC clamp") to isolate regions of a hybrid RNA as discrete folding units. In this work, we introduce new bait RNA constructs to (1) insulate the folding of individual components of the hybrid RNA with GC clamps and (2) express bait RNAs that do not encode their own intrinsic terminator. We find that short GC clamps (5 or 7 bp long) are more effective than a longer 13 bp GC clamp in the B3H assay. These new constructs increase the number of Hfq-sRNA and -5'UTR interactions that are detectable in the B3H system and improve the signal-to-noise ratio of many of these interactions. We therefore recommend the use of constructs containing short GC clamps for the expression of future B3H bait RNAs. With these new constructs, a broader range of RNA-protein interactions are detectable in the B3H assay, expanding the utility and impact of this genetic tool as a platform to search for and interrogate mechanisms of additional RNA-protein interactions.