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Probing Hfq:RNA Interactions with Hydroxyl Radical and RNase Footprinting

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

Key words

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

Notes

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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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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