title: Probing Hfq:RNA interactions with hydroxyl radical and RNase footprinting.
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 (32)P-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.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, 1151 Richmond St., London, ON, Canada, N6A 5C1.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Ellis
authors.forename: Ryan S
authors.initials: RS
authors.lastname: Trussler
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Ross
authors.forename: David B
authors.initials: DB
authors.lastname: Haniford
mesh_terms: D029968:Escherichia coli Proteins / Q000378:metabolism*; D035001:Host Factor 1 Protein / Q000378:metabolism*; D017665:Hydroxyl Radical / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012329:RNA, Bacterial / Q000378:metabolism*; D016601:RNA-Binding Proteins
chemical_list: D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D017665:Hydroxyl Radical