Probing Hfq:RNA Interactions with Hydroxyl Radical and RNase Footprinting
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 wordsRibonuclease 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).
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