Gravel, C. M.; Berry, K. E.

The bacterial three-hybrid (B3H) assay is a powerful genetic tool for detecting interactions between RNA and RNA-binding proteins (RBPs) and assessing the consequences of RBP mutations. This transcription-based system connects the strength of an RNA-protein interaction to the expression of a lacZ reporter gene in Escherichia coli cells. This in vivo approach allows researchers to dissect RNA-protein interactions within a cellular environment, bypassing the need for biochemical purification of RNAs or proteins. This chapter details a three-day protocol for generating quantitative B3H data. Since a significant challenge in B3H assays is RNA misfolding, we describe a recently optimized set of B3H constructs that mitigates this issue by isolating bait RNAs as discrete folding units.