Abstract
Structural biology plays a central role in gaining a full understanding of the myriad roles of RNA in biology. In recent years, innovative approaches in RNA purification and crystallographic methods have lead to the visualization of an increasing number of unique structures, providing new insights into its function at the atomic level. This article presents general protocols which have streamlined the process of obtaining a homogeneous sample of properly folded and active RNA in high concentrations that crystallizes well in the presence of a suitable heavy-atom for phasing. Of particular importance are approaches toward RNA crystallography that include exploring “construct space” as opposed to “condition space”. Moreover, development of a highly flexible method for experimentally phasing RNA crystals may open the door to a relatively simple means of solving these structures.
Andrea L. Edwards and Andrew D. Garst contributed equally to this work.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Pley, H.W., Flaherty, K.M. and McKay, D.B. (1994) Three-dimensional structure of a hammerhead ribozyme. Nature 372, 68–74.
Scott, W.G., Finch, J.T. and Klug, A. (1995) The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell 81, 991–1002.
Cate, J.H., Gooding, A.R., Podell, E., Zhou, K., Golden, B.L., Kundrot, C.E., Cech, T.R. and Doudna, J.A. (1996) Crystal structure of a group I ribozyme domain: principles of RNA packing. Science 273, 1678–1685.
Cate, J.H., Gooding, A.R., Podell, E., Zhou, K., Golden, B.L., Szewczak, A.A., Kundrot, C.E., Cech, T.R. and Doudna, J.A. (1996) RNA tertiary structure mediation by adenosine platforms. Science 273, 1696–1699.
Klein, D.J., Schmeing, T.M., Moore, P.B. and Steitz, T.A. (2001) The kink-turn: a new RNA secondary structure motif. EMBO J. 20, 4214–4221.
Kieft, J.S. and Batey, R.T. (2004) A general method for rapid and nondenaturing purification of RNAs. RNA 10, 988–995.
Griffiths-Jones, S., Moxon, S., Marshall, M., Khanna, A., Eddy, S.R. and Bateman, A. (2005) Rfam: annotating non-coding RNAs in complete genomes. Nucleic Acids Res. 33, D121–124.
Oubridge, C., Ito, N., Teo, C.H., Fearnley, I. and Nagai, K. (1995) Crystallisation of RNA-protein complexes. II. The application of protein engineering for crystallisation of the U1A protein-RNA complex. J. Mol. Biol. 249, 409–423.
Price, S.R., Ito, N., Oubridge, C., Avis, J.M. and Nagai, K. (1995) Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. J. Mol. Biol. 249, 398–408.
Batey, R.T., Sagar, M.B. and Doudna, J.A. (2001) Structural and energetic analysis of RNA recognition by a universally conserved protein from the signal recognition particle. J. Mol. Biol. 307, 229–246.
Scott, W.G., Finch, J.T., Grenfell, R., Fogg, J., Smith, T., Gait, M.J. and Klug, A. (1995) Rapid crystallization of chemically synthesized hammerhead RNAs using a double screening procedure. J. Mol. Biol. 250, 327–332.
Ferre-D'Amare, A.R. and Doudna, J.A. (2000) Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module. J. Mol. Biol. 295, 541–556.
Gilbert, S.D., Montange, R.K., Stoddard, C.D. and Batey, R.T. (2006) Structural studies of the purine and SAM binding riboswitches. Cold Spring Harb Symp. Quant. Biol. 71, 259–268.
Grundy, F.J. and Henkin, T.M. (1998) The S box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in gram-positive bacteria. Mol. Microbiol. 30, 737–749.
Winkler, W.C., Nahvi, A., Sudarsan, N., Barrick, J.E. and Breaker, R.R. (2003) An mRNA structure that controls gene expression by binding S-adenosylmethionine. Nat. Struct. Biol. 10, 701–707.
McPherson, A. (1999) Crystallization of Biological Macromolecules. Cold Spring Harbor Press, Cold Spring Harbor, NY.
Pley, H.W., Flaherty, K.M. and McKay, D.B. (1994) Model for an RNA tertiary interaction from the structure of an intermolecular complex between a GAAA tetraloop and an RNA helix. Nature 372, 111–113.
Doherty, E.A., Batey, R.T., Masquida, B. and Doudna, J.A. (2001) A universal mode of helix packing in RNA. Nat. Struct. Biol. 8, 339–343.
Nissen, P., Ippolito, J.A., Ban, N., Moore, P.B. and Steitz, T.A. (2001) RNA tertiary interactions in the large ribosomal subunit: the A-minor motif. Proc. Natl. Acad. Sci. U.S.A. 98, 4899–4903.
Montange, R.K. and Batey, R.T. (2006) Structure of the S-adenosylmethionine riboswitch regulatory mRNA element. Nature 441, 1172–1175.
Corbino, K.A., Barrick, J.E., Lim, J., Welz, R., Tucker, B.J., Puskarz, I., Mandal, M., Rudnick, N.D. and Breaker, R.R. (2005) Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria. Genome. Biol. 6, R70.
Gilbert, S.D., Rambo, R.P., Van Tyne, D., and Batey, R.T. (2008) Structure of the SAM-II riboswitch bound to S-adenosylmethionine. Nat. Struct. Biol. 15, 177–182.
Baugh, C., Grate, D. and Wilson, C. (2000) 2.8 A crystal structure of the malachite green aptamer. J. Mol. Biol. 301, 117–128.
Kieft, J.S., Zhou, K., Grech, A., Jubin, R. and Doudna, J.A. (2002) Crystal structure of an RNA tertiary domain essential to HCV IRES-mediated translation initiation. Nat. Struct. Biol. 9, 370–374.
Carrasco, N., Buzin, Y., Tyson, E., Halpert, E. and Huang, Z. (2004) Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion. Nucleic Acids Res. 32, 1638–1646.
Hobartner, C., Rieder, R., Kreutz, C., Puffer, B., Lang, K., Polonskaia, A., Serganov, A. and Micura, R. (2005) Syntheses of RNAs with up to 100 nucleotides containing site-specific 2'-methylseleno labels for use in X-ray crystallography. J. Am. Chem. Soc. 127, 12035–12045.
Golden, B.L. and Kundrot, C.E. (2003) RNA crystallization. J. Struct. Biol. 142, 98–107.
Keel, A.Y., Rambo, R.P., Batey, R.T. and Kieft, J.S. (2007) A general strategy to solve the phase problem in RNA crystallography. Structure 15, 761–772.
Ferre-D'Amare, A.R. and Doudna, J.A. (1996) Use of cis- and trans-ribozymes to remove 5' and 3' heterogeneities from milligrams of in vitro transcribed RNA. Nucleic Acids Res. 24, 977–978.
Batey, R.T. and Kieft, J.S. (2007) Improved native affinity purification of RNA. RNA 13, 1384–1389.
Grodberg, J. and Dunn, J.J. (1988) ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J. Bacteriol. 170, 1245–1253.
Zawadzki, V. and Gross, H.J. (1991) Rapid and simple purification of T7 RNA polymerase. Nucleic Acids Res. 19, 1948.
Li, Y., Wang, E. and Wang, Y. (1999) A modified procedure for fast purification of T7 RNA polymerase. Protein. Expr. Purif. 16, 355–358.
Ichetovkin, I.E., Abramochkin, G. and Shrader, T.E. (1997) Substrate recognition by the leucyl/phenylalanyl-tRNA-protein transferase. Conservation within the enzyme family and localization to the trypsin-resistant domain. J. Biol. Chem. 272, 33009–33014.
Pleiss, J.A., Derrick, M.L. and Uhlenbeck, O.C. (1998) T7 RNA polymerase produces 5' end heterogeneity during in vitro transcription from certain templates. RNA 4, 1313–1317.
Golden, B. L. (2007) Preparation and crystallization of RNA. Methods. Mol. Biol. 363, 239–257.
Kim, I., McKenna, S.A., Viani Puglisi, E. and Puglisi, J.D. (2007) Rapid purification of RNAs using fast performance liquid chromatography (FPLC). RNA 13, 289–294.
Bergfors, T. (2007) Screening and optimization methods for nonautomated crystallization laboratories. Methods Mol. Biol. 363, 131–151.
McCarthy, T.J., Plog, M.A., Floy, S.A., Jansen, J.A., Soukup, J.K. and Soukup, G.A. (2005) Ligand requirements for glmS ribozyme self-cleavage. Chem. Biol. 12, 1221–1226.
Roth, A., Nahvi, A., Lee, M., Jona, I. and Breaker, R.R. (2006) Characteristics of the glmS ribozyme suggest only structural roles for divalent metal ions. RNA 12, 607–619.
Acknowledgments
The contents of this work comprise much of the collected wisdom of a number of colleagues and members of the Batey laboratory. In particular, we would like to thank Jeffrey Kieft of the C.U. Health Sciences Center who has been instrumental in developing many of the ideas presented in this article. This work was made possible by a Research Scholar Grant from the American Cancer Society and support from the National Institutes of Health to R.T.B.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Edwards, A.L., Garst, A.D., Batey, R.T. (2009). Determining Structures of RNA Aptamers and Riboswitches by X-Ray Crystallography. In: Mayer, G. (eds) Nucleic Acid and Peptide Aptamers. Methods in Molecular Biology™, vol 535. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-557-2_9
Download citation
DOI: https://doi.org/10.1007/978-1-59745-557-2_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-89-3
Online ISBN: 978-1-59745-557-2
eBook Packages: Springer Protocols