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 identifi cation 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 proteindirected 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.
CITATION STYLE
Ellis, M. J., Trussler, R. S., Ross, J. A., & Haniford, D. B. (2015). Probing Hfq:RNA interactions with hydroxyl radical and rnase footprinting. Methods in Molecular Biology, 1259, 403–415. https://doi.org/10.1007/978-1-4939-2214-7_24
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