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

Nature Protocols volume 17, pages 941–961 (2022)Cite this article

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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 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.

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Fig. 1: General schematic of bacterial two- and three-hybrid systems.
Fig. 2: Plasmid maps of vectors used in B3H experiments highlighting key restriction sites used for cloning, promoters driving the expression of hybrid components and antibiotic resistance conferred by each plasmid.
Fig. 3: Flowchart depicting the process of a forward genetic screen and counter screen using a pPrey mutagenesis library (Steps 30–43).
Fig. 4: Example B3H data.

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Acknowledgements

We thank members of the Berry lab for discussion and suggestions and A. Hochschild, P. Deighan and D. Heller for discussions and advice in establishing these protocols. This work was supported with funding from the National Institutes of Health [R15GM135878], the Camille and Henry Dreyfus Foundation, the Henry R. Luce Foundation and Mount Holyoke College.

Author information

Author notes

  1. Oliver M. Stockert

    Present address: Medical Scientist Training Program, University of Minnesota Medical School, Minneapolis, MN, USA

  2. These authors contributed equally: Oliver M. Stockert, Chandra M. Gravel.

Authors and Affiliations

  1. Program in Biochemistry, Mount Holyoke College, South Hadley, MA, USA

    Oliver M. Stockert, Chandra M. Gravel & Katherine E. Berry

  2. Department of Chemistry, Mount Holyoke College, South Hadley, MA, USA

    Chandra M. Gravel & Katherine E. Berry

Authors
  1. Oliver M. Stockert
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Contributions

O.M.S., C.M.G. and K.E.B. organized, wrote and edited the manuscript. C.M.G. performed B3H experiments. O.M.S. and K.E.B. made figures.

Corresponding author

Correspondence to Katherine E. Berry.

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The authors declare no competing interests.

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Nature Protocols thanks Simon Dove, Michelle Meyer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Berry, K. E. & Hochschild, A. Nucleic Acids Res. 46, e12 (2018): https://doi.org/10.1093/nar/gkx1086

Wang C. D. et al. RNA 27, 513–526 (2021): https://rnajournal.cshlp.org/content/27/4/513.full

Pandey S. et al. Nucleic Acids Res. 48, 4507–4520 (2020): https://doi.org/10.1093/nar/gkaa144

Source data

Source Data Fig. 4a,b

Unprocessed photographs.

Source Data Fig. 4c,d,e

Statistical source data (raw beta-galactosidase data).

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Stockert, O.M., Gravel, C.M. & Berry, K.E. A bacterial three-hybrid assay for forward and reverse genetic analysis of RNA–protein interactions. Nat Protoc 17, 941–961 (2022). https://doi.org/10.1038/s41596-021-00657-4

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