Improved constructs for bait RNA display in a bacterial three-hybrid assay.

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 cl

SUBMITTER: Nguyen LD 

PROVIDER: S-EPMC11782564 | biostudies-literature | 2025 Jan

REPOSITORIES: biostudies-literature