Abstract
This chapter describes the preparation of NMR quantities of RNA purified to single-nucleotide resolution for protein–RNA interaction studies. The protocol is easily modified to make nucleotide-specific isotopically labeled RNAs or uniformly labeled RNA fragments for ligation to generate segmentally labeled RNAs.
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References
Autexier, C., and Triki, I. (1999) Tetrahymena telomerase ribonucleoprotein RNA–protein interactions. Nucl. Acids Res. 27, 2227–2234.
Bachand, F., Triki, I., and Autexier, C. (2001) Human telomerase RNA–protein interactions. Nucl. Acids Res. 29, 3385–3393.
Greider, C. W., and Blackburn, E. H. (1987) The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell 51, 887–898.
Staley, J. P., and Woolford, J. L., Jr. (2009) Assembly of ribosomes and spliceosomes: complex ribonucleoprotein machines. Curr. Opin. Cell Biol. 21, 109–118.
Ban, N., Nissen, P., Hansen, J., Moore, P. B., and Steitz, T. A. (2000) The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289, 905–920.
Cech, T. R. (2000) Structural biology. The ribosome is a ribozyme, Science 289, 878–879.
Schluenzen, F., Tocilj, A., Zarivach, R., Harms, J., Gluehmann, M., Janell, D., Bashan, A., Bartels, H., Agmon, I., Franceschi, F., and Yonath, A. (2000) Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution. Cell 102, 615–623.
Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Jr., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Structure of the 30S ribosomal subunit. Nature 407, 327–339.
Ule, J. (2008) Ribonucleoprotein complexes in neurologic diseases. Current Opinion in Neurobiology 18, 516–523.
Whittaker, J. W. (2007) Selective isotopic labeling of recombinant proteins using amino acid auxotroph strains. Methods. Mol. Biol. 389, 175–188.
Cowburn, D., Shekhtman, A., Xu, R., Ottesen, J. J., and Muir, T. W. (2004) Segmental isotopic labeling for structural biological applications of NMR. Methods Mol. Biol. 278, 47–56.
Liu, D., Xu, R., and Cowburn, D. (2009) Segmental isotopic labeling of proteins for nuclear magnetic resonance. Methods Enzymol. 462, 151–175.
Lu, K., Miyazaki, Y., and Summers, M. F. (2010) Isotope labeling strategies for NMR studies of RNA. J. Biomol. NMR 46, 113–125.
Nelissen, F. H., van Gammeren, A. J., Tessari, M., Girard, F. C., Heus, H. A., and Wijmenga, S. S. (2008) Multiple segmental and selective isotope labeling of large RNA for NMR structural studies. Nucl. Acids Res. 36, e89.
Peterson, R. D., Theimer, C. A., Wu, H., and Feigon, J. (2004) New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA–protein complexes. J. Biomol. NMR 28, 59–67.
Milligan, J. F., Groebe, D. R., Witherell, G. W., and Uhlenbeck, O. C. (1987) Oligoribo-nucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucl. Acids Res. 15, 8783–8798.
Cazenave, C., and Uhlenbeck, O. C. (1994) RNA template-directed RNA synthesis by T7 RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 91, 6972–6976.
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.
Triana-Alonso, F. J., Dabrowski, M., Wadzack, J., and Nierhaus, K. H. (1995) Self-coded 3′-extension of run-off transcripts produces aberrant products during in vitro transcription with T7 RNA polymerase. J. Biol. Chem. 270, 6298–6307.
Kao, C., Rudisser, S., and Zheng, M. (2001) A simple and efficient method to transcribe RNAs with reduced 3′ heterogeneity. Methods 23, 201–205.
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.
Nacheva, G. A., and Berzal-Herranz, A. (2003) Preventing nondesired RNA-primed RNA extension catalyzed by T7 RNA polymerase. Eur. J. Biochem./FEBS 270, 1458–1465.
Cunningham, P. R., and Ofengand, J. (1990) Use of inorganic pyrophosphatase to improve the yield of in vitro transcription reactions catalyzed by T7 RNA polymerase. BioTechniques 9, 713–714.
Gardner, K. H., and Kay, L. E. (1998) The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins. Ann. Rev. Biophys. Biomol. Struct. 27, 357–406.
Marley, J., Lu, M., and Bracken, C. (2001) A method for efficient isotopic labeling of recombinant proteins. J. Biomol. NMR 20, 71–75.
Wu, H., Finger, L. D., and Feigon, J. (2005) Structure determination of protein/RNA complexes by NMR. Methods Enzymol. 394, 525–545.
Marenchino, M., Armbruster, D. W., and Hennig, M. (2009) Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev. Protein Expression and Purification 63, 112–119.
Khanna, M., Wu, H., Johansson, C., Caizergues-Ferrer, M., and Feigon, J. (2006) Structural study of the H/ACA snoRNP components Nop10p and the 3′ hairpin of U65 snoRNA. RNA 12, 40–52.
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Theimer, C.A., Smith, N.L., Khanna, M. (2012). NMR Studies of Protein–RNA Interactions. In: Shekhtman, A., Burz, D. (eds) Protein NMR Techniques. Methods in Molecular Biology, vol 831. Humana Press. https://doi.org/10.1007/978-1-61779-480-3_12
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DOI: https://doi.org/10.1007/978-1-61779-480-3_12
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