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 techn...
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Published in | Methods in molecular biology (Clifton, N.J.) Vol. 1259; p. 403 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
2015
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Subjects | |
Online Access | Get more information |
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Summary: | 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. |
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ISSN: | 1940-6029 |
DOI: | 10.1007/978-1-4939-2214-7_24 |