A bacterial three-hybrid assay for forward and reverse genetic analysis of RNA-protein interactions

This protocol describes a three-hybrid system to analyze RNA-protein interactions in live bacteria and a complementary forward genetics screen to identify protein and RNA mutants with desired phenotypes. This protocol describes a bacterial three-hybrid (B3H) assay, an in vivo system that reports on RNA-protein interactions and can be implemented in both forward and reverse genetic experiments. The B3H assay connects the strength of an RNA-protein interaction inside of living Escherichia coli cells to the transcription of a reporter gene (here, lacZ). We present protocols to (1) insert RNA and protein sequences into appropriate vectors for B3H experiments, (2) detect putative RNA-protein interactions with both qualitative and quantitative readouts and (3) carry out forward genetic mutagenesis screens. The B3H assay builds on a well-established bacterial two-hybrid system for genetic analyses. As a result, protein-protein interactions can be assessed in tandem with RNA interactions with a bacterial two-hybrid assay to ensure that protein variants maintain their functionality. The B3H system is a powerful complement to traditional biochemical methods for dissecting RNA-protein interaction mechanisms: RNAs and proteins of interest do not need to be purified, and their interactions can be assessed under native conditions inside of a living bacterial cell. Once cloning has been completed, an assay can be completed in under a week and a screen in 1-2 weeks.

Automated summary

This protocol introduces a three-hybrid system and a forward genetics screen to analyze RNA-protein interactions in live bacteria. The system connects the strength of an RNA-protein interaction to the transcription of a reporter gene in Escherichia coli cells. It provides a powerful complement to traditional biochemical methods for studying RNA-protein interactions.