ArticleNucleic acids research2018
A bacterial three-hybrid assay detects Escherichia coli Hfq-sRNA interactions in vivo.
Article in Nucleic acids research, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed, 18 citations in OpenAlex.
- Decoding RNA-protein interactions using high-throughput methods.RNA biology · 2026Review
- Molecular genetic characterization of bacterial KH-domain proteins.bioRxiv : the preprint server for biology · 2026Article
- Improved constructs for bait RNA display in a bacterial three-hybrid assay.Scientific reports · 2025Article
- Improved constructs for bait RNA display in a bacterial three-hybrid assay.bioRxiv : the preprint server for biology · 2024Article
- Generation of single-cysteinemicroPublication biology · 2024Article
- Biochemical and genetic dissection of the RNA-binding surface of the FinO domain ofRNA (New York, N.Y.) · 2023Article
- Biochemical and genetic dissection of the RNA-binding surface of the FinO domain ofbioRxiv : the preprint server for biology · 2023Article
- A translational repression reporter assay for the analysis of RNA-binding protein consensus sites.RNA biology · 2023Article
- Identification of host factors binding to dengue and Zika virus subgenomic RNA by efficient yeast three-hybrid screens of the human ORFeome.RNA biology · 2021Article
- Optimization of a bacterial three-hybrid assay through in vivo titration of an RNA-DNA adapter protein.RNA (New York, N.Y.) · 2021Article
- Determinants of RNA recognition by the FinO domain of the Escherichia coli ProQ protein.Nucleic acids research · 2020Article
- Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ.Nucleic acids research · 2020Article
- The binding of Class II sRNA MgrR to two different sites on matchmaker protein Hfq enables efficient competition for Hfq and annealing to regulated mRNAs.RNA (New York, N.Y.) · 2018Article
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
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.
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