Skip to main content
NCBI home page
RNA logoLink to RNA
. 1999 Sep;5(9):1158–1166. doi: 10.1017/s1355838299990830

A novel 5 displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA.

J C Macosko 1, M S Pio 1, I Tinoco Jr 1, Y K Shin 1
PMCID: PMC1369839  PMID: 10496217

Abstract

An RNA spin-labeling technique was developed using the well-characterized interaction between the HIV Rev peptide and the Rev response element (RRE) RNA as a model system. Spin-labeled RNA molecules were prepared by incorporating guanosine monophosphorothioate (GMPS) at the 5' end using T7 RNA polymerase and then covalently attaching a thiol-specific nitroxide spin label. Three different constructs of the RRE RNA were made by strategically displacing the 5' end within the native three-dimensional structure. Nitroxide-to-nitroxide distance measurements were made between the specifically bound RNA and peptide using electron paramagnetic resonance (EPR) spectroscopy. The dipolar EPR method can reliably measure distances up to 25 A, the calculation of which is derived from the 1/r3 dependence of the broadening of EPR lines in motionally frozen samples. This RNA-labeling technique, dubbed 5' displacement spin labeling, extends the usefulness of the dipolar EPR method developed for analysis of protein structure. The advantage of this technique is that it is applicable to large RNA systems such as the ribosome, which are difficult to study by other structural methods.

Full Text

The Full Text of this article is available as a PDF (460.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altenbach C., Greenhalgh D. A., Khorana H. G., Hubbell W. L. A collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: application to spin-labeled mutants of bacteriorhodopsin. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1667–1671. doi: 10.1073/pnas.91.5.1667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altenbach C., Marti T., Khorana H. G., Hubbell W. L. Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants. Science. 1990 Jun 1;248(4959):1088–1092. doi: 10.1126/science.2160734. [DOI] [PubMed] [Google Scholar]
  3. Bartel D. P., Zapp M. L., Green M. R., Szostak J. W. HIV-1 Rev regulation involves recognition of non-Watson-Crick base pairs in viral RNA. Cell. 1991 Nov 1;67(3):529–536. doi: 10.1016/0092-8674(91)90527-6. [DOI] [PubMed] [Google Scholar]
  4. Battiste J. L., Mao H., Rao N. S., Tan R., Muhandiram D. R., Kay L. E., Frankel A. D., Williamson J. R. Alpha helix-RNA major groove recognition in an HIV-1 rev peptide-RRE RNA complex. Science. 1996 Sep 13;273(5281):1547–1551. doi: 10.1126/science.273.5281.1547. [DOI] [PubMed] [Google Scholar]
  5. Battiste J. L., Tan R., Frankel A. D., Williamson J. R. Assignment and modeling of the Rev Response Element RNA bound to a Rev peptide using 13C-heteronuclear NMR. J Biomol NMR. 1995 Dec;6(4):375–389. doi: 10.1007/BF00197637. [DOI] [PubMed] [Google Scholar]
  6. Chang K. Y., Varani G. Nucleic acids structure and recognition. Nat Struct Biol. 1997 Oct;4 (Suppl):854–858. [PubMed] [Google Scholar]
  7. Emerman M., Malim M. H. HIV-1 regulatory/accessory genes: keys to unraveling viral and host cell biology. Science. 1998 Jun 19;280(5371):1880–1884. doi: 10.1126/science.280.5371.1880. [DOI] [PubMed] [Google Scholar]
  8. Fiori W. R., Millhauser G. L. Exploring the peptide 3(10)-helix reversible alpha-helix equilibrium with double label electron spin resonance. Biopolymers. 1995;37(4):243–250. doi: 10.1002/bip.360370403. [DOI] [PubMed] [Google Scholar]
  9. Hall J. A., Thorgeirsson T. E., Liu J., Shin Y. K., Nikaido H. Two modes of ligand binding in maltose-binding protein of Escherichia coli. Electron paramagnetic resonance study of ligand-induced global conformational changes by site-directed spin labeling. J Biol Chem. 1997 Jul 11;272(28):17610–17614. doi: 10.1074/jbc.272.28.17610. [DOI] [PubMed] [Google Scholar]
  10. Harris M. E., Kazantsev A. V., Chen J. L., Pace N. R. Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking. RNA. 1997 Jun;3(6):561–576. [PMC free article] [PubMed] [Google Scholar]
  11. Holbrook S. R., Kim S. H. RNA crystallography. Biopolymers. 1997;44(1):3–21. doi: 10.1002/(SICI)1097-0282(1997)44:1<3::AID-BIP2>3.0.CO;2-Z. [DOI] [PubMed] [Google Scholar]
  12. Hubbell W. L., Gross A., Langen R., Lietzow M. A. Recent advances in site-directed spin labeling of proteins. Curr Opin Struct Biol. 1998 Oct;8(5):649–656. doi: 10.1016/s0959-440x(98)80158-9. [DOI] [PubMed] [Google Scholar]
  13. Hwang I., Thorgeirsson T., Lee J., Kustu S., Shin Y. K. Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC. Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):4880–4885. doi: 10.1073/pnas.96.9.4880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Joseph S., Weiser B., Noller H. F. Mapping the inside of the ribosome with an RNA helical ruler. Science. 1997 Nov 7;278(5340):1093–1098. doi: 10.1126/science.278.5340.1093. [DOI] [PubMed] [Google Scholar]
  15. King D. S., Fields C. G., Fields G. B. A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Int J Pept Protein Res. 1990 Sep;36(3):255–266. doi: 10.1111/j.1399-3011.1990.tb00976.x. [DOI] [PubMed] [Google Scholar]
  16. Macosko J. C., Kim C. H., Shin Y. K. The membrane topology of the fusion peptide region of influenza hemagglutinin determined by spin-labeling EPR. J Mol Biol. 1997 Apr 18;267(5):1139–1148. doi: 10.1006/jmbi.1997.0931. [DOI] [PubMed] [Google Scholar]
  17. Malim M. H., Hauber J., Le S. Y., Maizel J. V., Cullen B. R. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989 Mar 16;338(6212):254–257. doi: 10.1038/338254a0. [DOI] [PubMed] [Google Scholar]
  18. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ottemann K. M., Thorgeirsson T. E., Kolodziej A. F., Shin Y. K., Koshland D. E., Jr Direct measurement of small ligand-induced conformational changes in the aspartate chemoreceptor using EPR. Biochemistry. 1998 May 19;37(20):7062–7069. doi: 10.1021/bi980305e. [DOI] [PubMed] [Google Scholar]
  20. Poirier M. A., Xiao W., Macosko J. C., Chan C., Shin Y. K., Bennett M. K. The synaptic SNARE complex is a parallel four-stranded helical bundle. Nat Struct Biol. 1998 Sep;5(9):765–769. doi: 10.1038/1799. [DOI] [PubMed] [Google Scholar]
  21. Rabenstein M. D., Shin Y. K. Determination of the distance between two spin labels attached to a macromolecule. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8239–8243. doi: 10.1073/pnas.92.18.8239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rabenstein M. D., Shin Y. K. HIV-1 gp41 tertiary structure studied by EPR spectroscopy. Biochemistry. 1996 Nov 5;35(44):13922–13928. doi: 10.1021/bi961743t. [DOI] [PubMed] [Google Scholar]
  23. Shin Y. K. K+ channel gating mechanism proposed using EPR. Nat Struct Biol. 1998 Jun;5(6):418–420. doi: 10.1038/nsb0698-418. [DOI] [PubMed] [Google Scholar]
  24. Tan R., Chen L., Buettner J. A., Hudson D., Frankel A. D. RNA recognition by an isolated alpha helix. Cell. 1993 Jun 4;73(5):1031–1040. doi: 10.1016/0092-8674(93)90280-4. [DOI] [PubMed] [Google Scholar]
  25. Thorgeirsson T. E., Xiao W., Brown L. S., Needleman R., Lanyi J. K., Shin Y. K. Transient channel-opening in bacteriorhodopsin: an EPR study. J Mol Biol. 1997 Nov 14;273(5):951–957. doi: 10.1006/jmbi.1997.1362. [DOI] [PubMed] [Google Scholar]
  26. Wyatt J. R., Chastain M., Puglisi J. D. Synthesis and purification of large amounts of RNA oligonucleotides. Biotechniques. 1991 Dec;11(6):764–769. [PubMed] [Google Scholar]
  27. Yu Y. G., Thorgeirsson T. E., Shin Y. K. Topology of an amphiphilic mitochondrial signal sequence in the membrane-inserted state: a spin labeling study. Biochemistry. 1994 Nov 29;33(47):14221–14226. doi: 10.1021/bi00251a034. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES