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Crystallographic Analysis of Small Ribozymes and Riboswitches

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Book cover Ribozymes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 848))

Abstract

Ribozymes and riboswitches are RNA motifs that accelerate biological reactions and regulate gene expression in response to metabolite recognition, respectively. These RNA molecules gain functionality via complex folding that cannot be predicted a priori, and thus requires high-resolution three-dimensional structure determination to locate key functional attributes. Herein, we present an overview of the methods used to determine small RNA structures with an emphasis on RNA preparation, crystallization, and structure refinement. We draw upon examples from our own research in the analysis of the leadzyme ribozyme, the hairpin ribozyme, a class I preQ1 riboswitch, and variants of a larger class II preQ1 riboswitch. The methods presented provide a guide for comparable investigations of noncoding RNA molecules including a 48-solution, “first choice” RNA crystal screen compiled from our prior successes with commercially available screens.

Geoffrey M. Lippa; Joseph A. Liberman; Jermaine L. Jenkins contributed equally to this manuscript.

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Acknowledgments

We thank Profs. Harold C. Smith and Clara L. Kielkopf for sharing their expertise on RNA. We thank Jason Salter for assistance with diffraction analysis, as well as the staff of MacCHESS and SSRL for help with X-ray data collection. This work was supported in part by NIH grants GM063162 and RR026501 to J.E.W. MacCHESS is supported by NSF award DMR-0225180 and NIH/NCRR award RR01646SSRL. SSRL is operated by Stanford on behalf of the U.S. DOE. The SSRL Structural Molecular Biology Program is supported by the DOE, and by NIH/NCRR and NIGMS.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY, USA

    Geoffrey M. Lippa, Joseph A. Liberman, Jermaine L. Jenkins, Jolanta Krucinska, Mohammad Salim & Joseph E. Wedekind

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  1. Geoffrey M. Lippa
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  2. Joseph A. Liberman
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  3. Jermaine L. Jenkins
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  4. Jolanta Krucinska
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  5. Mohammad Salim
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  6. Joseph E. Wedekind
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Corresponding author

Correspondence to Joseph E. Wedekind .

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Editors and Affiliations

  1. Department of Chemistry, Universität Konstanz, Universitätsstr. 10, Konstanz, 78457, Germany

    Jörg S. Hartig

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Lippa, G.M., Liberman, J.A., Jenkins, J.L., Krucinska, J., Salim, M., Wedekind, J.E. (2012). Crystallographic Analysis of Small Ribozymes and Riboswitches. In: Hartig, J. (eds) Ribozymes. Methods in Molecular Biology, vol 848. Humana Press. https://doi.org/10.1007/978-1-61779-545-9_11

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  • DOI: https://doi.org/10.1007/978-1-61779-545-9_11

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-544-2

  • Online ISBN: 978-1-61779-545-9

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