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

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Ribozymes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2167))

Abstract

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.

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Change history

  • 13 October 2020

    Owing to an oversight, the words “Reverse” and “Cleaved” in Subheading 2.3.3 of Chapter 9 were spelt incorrectly in the book.

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Acknowledgments

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. Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, OH, USA

    Stella M. Lai & Venkat Gopalan

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  1. Stella M. Lai
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  2. Venkat Gopalan
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Correspondence to Venkat Gopalan .

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Editors and Affiliations

  1. Department of Microbiology and Immunology, Lady Davis Institute for Medical Research, Montréal, QC, Canada

    Robert J Scarborough

  2. Department of Microbiology and Immunology, Lady Davis Institute for Medical Research, Montréal, QC, Canada

    Anne Gatignol

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Lai, S.M., Gopalan, V. (2021). Using an L7Ae-Tethered, Hydroxyl Radical-Mediated Footprinting Strategy to Identify and Validate Kink-Turns in RNAs. In: Scarborough, R.J., Gatignol, A. (eds) Ribozymes. Methods in Molecular Biology, vol 2167. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0716-9_9

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  • DOI: https://doi.org/10.1007/978-1-0716-0716-9_9

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