Abstract
The field of RNA structure has exploded in recent years, in part owing to advances in crystallography of RNA molecules. This phenomenon can largely be attributed to the development of three modern methods: (1) large-scale in vitro RNA synthesis, (2) cryocrystallography, and (3) high-intensity synchrotron beamlines. Milligram quantities of RNA can be routinely synthesized using either chemical or enzymatic syntheses, making it feasible to carry out routine crystallization experiments on RNA. This has allowed crystals of RNA to be readily obtained. Generally, RNA crystals tend to be susceptible to radiation damage and to diffract X-rays more weakly than their protein counterparts. However, cryocrystallography and the high-intensity X-ray sources have overcome many of the difficulties involved in solving crystal structures of RNA. As a result of these advances, we now have a database of RNA structures that span from simple duplexes and hairpins to complex ribozymes and ribosomes. The protocols presented here describe methods to synthesize, purify, crystallize, and derivatize RNA for use in crystallographic studies.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Anderson, A. C., Earp, B. E., and Frederick, C. A. (1996) Sequence variation as a strategy for crystallizing RNA motifs. J. Mol. Biol. 259, 696–703.
Correll, C. C., Wool, I. G., and Munishkin, A. (1999) The two faces of the Escherichia coli 23 S rRNA sarcin/ricin domain: the structure at 1.11 Å resolution. J. Mol. Biol. 292, 275–287.
Golden, B. L., Podell, E. R., Gooding, A. R., and Cech, T. R. (1997) Crystals by design: a strategy for crystallization of a ribozyme derived from the Tetrahymena group I intron. J. Mol. Biol. 270, 711–723.
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.
Scott, W. G., Finch, J. T., Grenfell, R., et al. (1995) Rapid crystallization of chemically synthesized hammerhead RNAs using a double screening procedure. J. Mol. Biol. 250, 327–332.
Hoggan, D. B., Chao, J. A., Prasad, G. S., Stout, C. D., and Williamson, J. R. (2003) Combinatorial crystallization of an RNA-protein complex. Acta Crystallogr. D. Biol. Crystallogr. 59, 466–473.
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.
Ferré-D’Amaré, 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.
Shields, T. P., Mollova, E., Ste Marie, L., Hansen, M. R., and Pardi, A. (1999) High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme. RNA 5, 1259–1267.
Grosshans, C. A. and Cech, T. R. (1991) A hammerhead ribozyme allows synthesis of a new form of the tetrahymena ribozyme homogeneous in length with a 3′ end blocked for transesterification. Nucleic Acids Res. 19, 3875–3880.
Milligan, J. F. and Uhlenbeck, O. C. (1989) Synthesis of small RNAs using T7 RNA polymerase. Meth. Enzymol. 180, 51–62.
Kao, C., Zheng, M., and Rudisser, S. (1999) A simple and efficient method to reduce nontemplated nucleotide addition at the 3 terminus of RNAs transcribed by T7 RNA polymerase. RNA 5, 1268–1272.
Davanloo, P., Rosenberg, A. H., Dunn, J. J., and Studier, F. W. (1984) Cloning and expression of the gene for bacteriophage-T7 RNA-polymerase. Proc. Natl. Acad. Sci. USA 81, 2035–2039.
Li, Y., Wang, E., and Wang, Y. (1999) A modified procedure for fast purification of T7 RNA polymerase. Protein Expr. Purif. 16, 355–358.
Chen, C. J., Liu, Z. J., Rose, J. P., and Wang, B. C. (1999) Low-salt crystallization of T7 RNA polymerase: a first step towards the transcription bubble complex. Acta Crystallogr. D. Biol. Crystallogr. 55, 1188–1192.
Uhlenbeck, O. C. (1995) Keeping RNA happy. RNA 1, 4–6.
Doudna, J. A., Grosshans, C., Gooding, A., and Kundrot, C. E. (1993) Crystallization of ribozymes and small RNA motifs by a sparse matrix approach. Proc. Natl. Acad. Sci. USA 90, 7829–7833.
Berger, I., Kang, C. H., Sinha, N., Wolters, M., and Rich, A. (1996) A highly efficient 24-condition matrix for the crystallization of nucleic acid fragments. Acta Crystallogr. D. Biol. Crystallogr. 52, 465–468.
Cate, J. H. and Doudna, J. A. (1996) Metal-binding sites in the major groove of a large ribozyme domain. Structure 4, 1221–1229.
Cruse, W., Saludjian, P., Neuman, A., and Prange, T. (2001) Destabilizing effect of a fluorouracil extra base in a hybrid RNA duplex compared with bromo and chloro analogues. Acta Crystallogr. D. Biol. Crystallogr. 57, 1609–1613.
Basu, S., Rambo, R. P., Strauss-Soukup, J., et al. (1998) A specific monovalent metal ion integral to the AA platform of the RNA tetraloop receptor. Nat. Struct. Biol. 5, 986–992.
Ennifar, E., Carpentier, P., Ferrer, J. L., Walter, P., and Dumas, P. (2002) X-ray-induced debromination of nucleic acids at the Br K absorption edge and implications for MAD phasing. Acta Crystallogr. D. Biol. Crystallogr. 58, 1262–1268.
Carrasco, N., Ginsburg, D., Du, Q., and Huang, Z. (2001) Synthesis of selenium-derivatized nucleosides and oligonucleotides for X-ray crystallography. Nucleosides Nucleotides Nucleic Acids 20, 1723–1734.
Yang, W. and Steitz, T. A. (1995) Crystal structure of the site-specific recombinase gamma delta resolvase complexed with a 34 bp cleavage site. Cell 82, 193–207.
Horvath, M. P., Schweiker, V. L., Bevilacqua, J. M., Ruggles, J. A., and Schultz, S. C. (1998) Crystal structure of the Oxytricha nova telomere end binding protein complexed with single strand DNA. Cell 95, 963–974.
Slim, G. and Gait, M. J. (1991) Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. Nucleic Acids Res. 19, 1183–1188.
Moore, M. J. and Query, C. C. (2000) Joining of RNAs by splinted ligation. Meth. Enzymol. 317, 109–123.
Golden, B. L., Gooding, A. R., Podell, E. R., and Cech, T. R. (1996) X-ray crystallography of large RNAs: heavy-atom derivatives by RNA engineering. RNA 2, 1295–1305.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Golden, B.L. (2007). Preparation and Crystallization of RNA. In: Walker, J.M., Doublié, S. (eds) Macromolecular Crystallography Protocols. Methods in Molecular Biology, vol 363. Humana Press. https://doi.org/10.1007/978-1-59745-209-0_12
Download citation
DOI: https://doi.org/10.1007/978-1-59745-209-0_12
Publisher Name: Humana Press
Print ISBN: 978-1-58829-292-6
Online ISBN: 978-1-59745-209-0
eBook Packages: Springer Protocols