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RNA Remodeling Proteins pp 403–415Cite as

Probing Hfq:RNA Interactions with Hydroxyl Radical and RNase Footprinting

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1259))

Abstract

RNA footprinting and structure probing techniques are used to characterize the interaction between RNA-binding proteins and RNAs in vitro. Hydroxyl radical footprinting results in the identification of protein binding site(s) in an RNA. Ribonuclease (RNase) structure probing is a complementary technique that also provides information about protein binding sites, as well as RNA structure and possible protein-directed RNA remodeling. Here we provide a comprehensive protocol for studying the interaction between Hfq and an mRNA or sRNA of interest using a combination of RNase A, T1, and V1 as well as hydroxyl radical footprinting techniques. Detailed protocols for in vitro synthesis of 32P-labeled RNA; formation of Hfq:RNA binary complex(es), RNase, and hydroxyl radical footprinting; preparation and running of sequencing gels; and data analysis are provided.

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Acknowledgments

We thank Brian Munshaw for helpful discussions regarding the protocols described here. This work was supported by a grant to D.B.H. from the Canadian Institutes of Health Research (CIHR; MOP 11281).

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Authors and Affiliations

  1. Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, 1151 Richmond St., London, ON, Canada, N6A 5C1

    Michael J. Ellis, Ryan S. Trussler, Joseph A. Ross & David B. Haniford

Authors
  1. Michael J. Ellis
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  2. Ryan S. Trussler
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  3. Joseph A. Ross
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  4. David B. Haniford
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Corresponding author

Correspondence to David B. Haniford .

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Editors and Affiliations

  1. CNRS Centre de Biophysique Moléculaire, Orléans, France

    Marc Boudvillain

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Ellis, M.J., Trussler, R.S., Ross, J.A., Haniford, D.B. (2015). Probing Hfq:RNA Interactions with Hydroxyl Radical and RNase Footprinting. In: Boudvillain, M. (eds) RNA Remodeling Proteins. Methods in Molecular Biology, vol 1259. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2214-7_24

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  • DOI: https://doi.org/10.1007/978-1-4939-2214-7_24

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2213-0

  • Online ISBN: 978-1-4939-2214-7

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