Ribozymes pp 147-169 | Cite as

Using an L7Ae-Tethered, Hydroxyl Radical-Mediated Footprinting Strategy to Identify and Validate Kink-Turns in RNAs

  • Stella M. Lai
  • Venkat GopalanEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2167)


Kink-turns are important RNA structural modules that facilitate long-range tertiary interactions and form binding sites for members of the L7Ae family of proteins. Present in a wide variety of functional RNAs, kink-turns play key organizational roles in many RNA-based cellular processes, including translation, modification, and tRNA biogenesis. It is important to determine the contribution of kink-turns to the overall architecture of resident RNAs, as these modules dictate ribonucleoprotein (RNP) assembly and function. This chapter describes a site-directed, hydroxyl radical-mediated footprinting strategy that utilizes L7Ae-tethered chemical nucleases to experimentally validate computationally identified kink-turns in any RNA and under a wide variety of conditions. The work plan described here uses the catalytic RNase P RNA as an example to provide a blueprint for using this footprinting method to map RNA–protein interactions in other RNP complexes.

Key words

hydroxyl radical footprinting L7Ae kink-turn RNA–protein interactions RNase P ribonucleoprotein complex 



We thank Lien Lai (The Ohio State University) for her initial efforts toward the development and optimization of the OH-mediated footprinting protocol described herein. We also gratefully acknowledge research support from the Behrman Research Fund (to V.G.) and National Institutes of Health (R01-GM120582 to V.G., Mark P. Foster, Julius B. Lucks, Michael G. Poirier, and Vicki H. Wysocki).


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Authors and Affiliations

  1. 1.Department of Chemistry and Biochemistry and Center for RNA BiologyThe Ohio State UniversityColumbusUSA

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