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

ABSTRACT: 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 ba

SUBMITTER: Stockert OM 

PROVIDER: S-EPMC10241318 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature