Berry, Katherine EHochschild, Ann2018-02-262018Berry, Katherine E., and Ann Hochschild. 2018. “A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo.” Nucleic Acids Research 46 (2): e12. doi:10.1093/nar/gkx1086. http://dx.doi.org/10.1093/nar/gkx1086.http://nrs.harvard.edu/urn-3:HUL.InstRepos:34868732Abstract The interaction of RNA molecules with proteins is a critical aspect of gene regulation across all domains of life. Here, we report the development of a bacterial three-hybrid (B3H) assay to genetically detect RNA–protein interactions. The basis for this three-hybrid assay is a transcription-based bacterial two-hybrid assay that has been used widely to detect and dissect protein–protein interactions. In the three-hybrid assay, a DNA-bound protein with a fused RNA-binding moiety (the coat protein of bacteriophage MS2 (MS2CP)) is used to recruit a hybrid RNA upstream of a test promoter. The hybrid RNA consists of a constant region that binds the tethered MS2CP and a variable region. Interaction between the variable region of the hybrid RNA and a target RNA-binding protein that is fused to a subunit of Escherichia coli RNA polymerase (RNAP) stabilizes the binding of RNAP to the test promoter, thereby activating transcription of a reporter gene. We demonstrate that this three-hybrid assay detects interaction between non-coding small RNAs (sRNAs) and the hexameric RNA chaperone Hfq from E. coli and enables the identification of Hfq mutants with sRNA-binding defects. Our findings suggest that this B3H assay will be broadly applicable for the study of RNA–protein interactions.en-USJournal Article2018-02-2610.1093/nar/gkx1086