Probing Hfq:RNA Interactions with Hydroxyl Radical and RNase Footprinting

  • Michael J. Ellis
  • Ryan S. Trussler
  • Joseph A. Ross
  • David B. HanifordEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)


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.

Key words

Ribonuclease footprinting Hydroxyl radical footprinting RNA–protein interactions RNA-binding proteins Hfq RNA structure Escherichia coli Sequencing gel In vitro transcription 



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).


  1. 1.
    Vogel J, Luisi BF (2011) Hfq and its constellation of RNA. Nat Rev Microbiol 9:578–589PubMedCrossRefGoogle Scholar
  2. 2.
    Storz G, Vogel J, Wassarman KM (2011) Regulation by small RNAs in bacteria: expanding frontiers. Mol Cell 43:880–891PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Valentin-Hansen P, Eriksen M, Udesen C (2004) MicroReview: the bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol Microbiol 51:1525–1533PubMedCrossRefGoogle Scholar
  4. 4.
    Brennan RG, Link TM (2007) Hfq structure, function and ligand binding. Curr Opin Microbiol 10:125–133PubMedCrossRefGoogle Scholar
  5. 5.
    Soper TJ, Doxzen K, Woodson SA (2011) Major role for mRNA binding and restructuring in sRNA recruitment by Hfq. RNA 17:1544–1550PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Geissmann TA, Touati D (2004) Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator. EMBO J 23:396–405PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Ross JA, Ellis MJ, Hossain S et al (2013) Hfq restructures RNA-IN and RNA-OUT and facilitates antisense pairing in the Tn10/IS10 system. RNA 19:670–684PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Ehresmann C, Baudin F, Mougel M et al (1987) Probing the structure of RNAs in solution. Nucleic Acids Res 15:9109–9128PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Chevalier C, Geissmann T, Helfer A-C et al (2009) Probing mRNA Structure and sRNA–mRNA Interactions in Bacteria Using Enzymes and Lead(II). Methods Mol Biol 540:215–232PubMedCrossRefGoogle Scholar
  10. 10.
    Rushizky GW, Knight CA, Sober HA (1961) Studies on the preferential specificity of pancreatic ribonuclease as deduced from partial digests. J Biol Chem 236:2732–2737PubMedGoogle Scholar
  11. 11.
    Pace CN, Heinemann U, Hahn U et al (1991) Ribonuclease T1: structure, function, and stability. Angew Chem Int Ed Engl 30:343–360CrossRefGoogle Scholar
  12. 12.
    Lowman HB, Draper DE (1986) On the recognition of helical RNA by cobra venom V1 nuclease. J Biol Chem 261:5396–5403PubMedGoogle Scholar
  13. 13.
    Sawadogo M, Roeder RG (1985) Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region. Cell 43:165–175PubMedCrossRefGoogle Scholar
  14. 14.
    Tullius TD, Dombroski BA, Churchill MEA et al (1987) [33] Hydroxyl radical footprinting: a high-resolution method for mapping protein-DNA contacts. In: Ray W (ed) Methods in Enzymology, vol 155. Academic, New York, pp 537–558Google Scholar
  15. 15.
    Powers T, Noller HF (1995) Hydroxyl radical footprinting of ribosomal proteins on 16S rRNA. RNA 1:194–209PubMedCentralPubMedGoogle Scholar
  16. 16.
    Fenton HJH (1894) LXXIII.-Oxidation of tartaric acid in presence of iron. J Chem Soc Trans 65:899–910CrossRefGoogle Scholar
  17. 17.
    Jain SS, Tullius TD (2008) Footprinting protein-DNA complexes using the hydroxyl radical. Nat Protoc 3:1092–1100PubMedCrossRefGoogle Scholar
  18. 18.
    Peng Y, Soper TJ, Woodson SA (2012) RNase footprinting of protein binding sites on an mRNA target of small RNAs. Methods Mol Biol 905:213–224PubMedGoogle Scholar
  19. 19.
    Zhang A, Wassarman KM, Ortega J et al (2002) The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs. Mol Cell 9:11–22PubMedCrossRefGoogle Scholar
  20. 20.
    Gagnon K, Maxwell ES (2011) Electrophoretic mobility shift assay for characterizing RNA–protein interaction. Methods Mol Biol 703:275–291PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Michael J. Ellis
    • 1
  • Ryan S. Trussler
    • 1
  • Joseph A. Ross
    • 1
  • David B. Haniford
    • 1
    Email author
  1. 1.Department of Biochemistry, Schulich School of Medicine & DentistryUniversity of Western OntarioLondonCanada

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