Probing Hfq:RNA interactions with hydroxyl radical and RNase footprinting

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1259; p. 403
• Main Authors: Ellis, Michael J, Trussler, Ryan S, Ross, Joseph A, Haniford, David B
• Format: Journal Article
• Language: English
• Published: United States 2015
• Subjects: Escherichia coli Proteins - metabolism; Host Factor 1 Protein - metabolism; Hydroxyl Radical - metabolism; Nucleic Acid Conformation; Protein Binding; RNA, Bacterial - metabolism; RNA-Binding Proteins
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-2214-7_24

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.