Advertisement

Journal of Biomolecular NMR

, Volume 35, Issue 4, pp 261–274 | Cite as

Alternate-site isotopic labeling of ribonucleotides for NMR studies of ribose conformational dynamics in RNA

  • James E. JohnsonJr
  • Kristine R. Julien
  • Charles G. Hoogstraten
Article

Abstract

Heteronuclear NMR spin relaxation studies of conformational dynamics are coming into increasing use to help understand the functions of ribozymes and other RNAs. Due to strong \(^{13}\hbox{C}--^{13}\hbox{C}\) magnetic interactions within the ribose ring, however, these studies have thus far largely been limited to 13C and 15N resonances on the nucleotide base side chains. We report here the application of the alternate-site 13C isotopic labeling scheme, pioneered by LeMaster for relaxation studies of amino acid side chains, to nucleic acid systems. We have used different strains of E. coli to prepare mononucleotides containing 13C label in one of two patterns: Either C1′ or C2′ in addition to C4′, termed (1′/2′,4′) labeling, or nearly complete labeling at the C2′ and C4′ sites only, termed (2′,4′) labeling. These patterns provide isolated \(^{13}\hbox{C}--^{1}\)H spin systems on the labeled carbon atoms and thus allow spin relaxation studies without interference from \(^{13}\hbox{C}--^{13}\hbox{C}\) scalar or dipolar coupling. Using relaxation studies of AMP dissolved in glycerol at varying temperature to produce systems with correlation times characteristic of different size RNAs, we demonstrate the removal of errors due to \(^{13}\hbox{C}--^{13}\hbox{C}\) interaction in T 1 measurements of larger nucleic acids and in T measurements in RNA molecules. By extending the applicability of spin relaxation measurements to backbone ribose groups, this technology should greatly improve the flexibility and completeness of NMR analyses of conformational dynamics in RNA.

Key words

alternate-site dynamics glucose-6-phosphate dehydrogenase isotope labeling ribose RNA 

Abbreviations

AMP

adenosine 5′-monophosphate

CMP

cytidine 5′-monophosphate

CPMG

Carr-Purcell-Meiboom-Gill

CSA

chemical shift anisotropy

G3P

glyceraldehyde-3-phosphate

G6P

glucose-6-phosphate

G6PDH

glucose-6-phosphate dehydrogenase

HSQC

heteronuclear single-quantum correlation

R5P

ribose-5-phosphate

rNMP

ribonucleoside 5′-monophosphate

TCA

tricarboxylic acid

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Notes

Acknowledgements

The authors are grateful to David LeMaster for helpful discussions, to John SantaLucia for comments on the manuscript, and to the Coli Genetic Stock Center (Yale) for bacterial strains. This work was supported by faculty startup funds and an Intramural Research Program Grant from Michigan State University and by the National Institutes of Health (GM-069742).

References

  1. Akke M. (2002). Curr. Opin. Struct. Biol. 12:642–647CrossRefGoogle Scholar
  2. Akke M., Fiala R., Jiang F., Patel D., Palmer A.G. III (1997). RNA 3:702–709Google Scholar
  3. al-Hashimi HM (2005). Chembiochem 6:1506–1519CrossRefGoogle Scholar
  4. Batey R.T., Battiste J.L., Williamson J.R. (1995). Methods Enzymol. 261:300–322Google Scholar
  5. Batey R.T., Inada M., Kujawinski E., Puglisi J.D., Williamson J.R. (1992). Nucl. Acids. Res. 20:4515–4523CrossRefGoogle Scholar
  6. Bax A., Davis D.G. (1985). J. Magn. Reson. 63:207–213Google Scholar
  7. Blad H., Reiter N.J., Abildgaard F., Markley J.L., Butcher S.E. (2005). J. Mol. Biol. 353:540–555CrossRefGoogle Scholar
  8. Boisbouvier J., Brutscher B., Simorre J.-P., Marion D. (1999). J. Biomolec. NMR 14:241–252CrossRefGoogle Scholar
  9. Boisbouvier J., Wu Z., Ono A., Kainosho M., Bax A. (2003). J. Biomolec. NMR 27:133–142CrossRefGoogle Scholar
  10. Borer P.N., LaPlante S.R., Anil Kumar, Zanatta N., Martin A., Hakkinen A., Levy G.C. (1994). Biochemistry 33:2441–2450CrossRefGoogle Scholar
  11. Bryce D.L., Grishaev A., Bax A. (2005). J. Am. Chem. Soc. 127:7387–7396CrossRefGoogle Scholar
  12. Castellani F., van Rossum B., Diehl A., Schubert M., Rehbein K., Oschkinat H. (2002). Nature 420:98–102CrossRefADSGoogle Scholar
  13. Castellani F., van Rossum B.J., Diehl A., Rehbein K., Oschkinat H. (2003). Biochemistry 42:11476–11483CrossRefGoogle Scholar
  14. Catoire L.J. (2004). J. Biomol. NMR 28:179–184CrossRefGoogle Scholar
  15. D’Souza V., Dey A., Habib D., Summers M.F. (2004). J. Mol. Biol. 337:427–442CrossRefGoogle Scholar
  16. Dayie K.T., Brodsky A.S., Williamson J.R. (2002). J. Mol. Biol. 317:263–278CrossRefGoogle Scholar
  17. Dayie K.T., Wagner G., Lefèvre J.F. (1996). Annu. Rev. Phys. Chem. 47:243–282CrossRefGoogle Scholar
  18. Duchardt E., Schwalbe H. (2005). J. Biomol. NMR. 32:295–308CrossRefGoogle Scholar
  19. Ebrahimi M., Rossi P., Rogers C., Harbison G.S. (2001). J. Magn. Reson. 150:1–9CrossRefADSGoogle Scholar
  20. Edwards J.S., Palsson B.O. (2000). Proc. Natl. Acad. Sci. USA 97:5528–5533CrossRefADSGoogle Scholar
  21. Fraenkel D.G. (1968). J. Bacteriol. 95:1267–1271Google Scholar
  22. Gaudin F., Chanteloup L., Thuong N.T., Lancelot G. (1997). Magn. Reson. Chem. 35:561–565CrossRefGoogle Scholar
  23. Gaudin F., Paquet F., Chanteloup L., Beau J.-M., Thuong N.T., Lancelot G. (1995). J. Biomolec. NMR 5:49–58CrossRefGoogle Scholar
  24. Hall K.B., Tang C. (1998). Biochemistry 37:9323–9332CrossRefGoogle Scholar
  25. Hatala P.J., Kallmerten J., Borer P.N. (2001). Nucleosides Nucleotides Nucleic Acids 20:1961–1973CrossRefGoogle Scholar
  26. Hines J.V., Landry S.M., Varani G., Tinoco I. (1994). J. Am. Chem. Soc. 116:5823–5831CrossRefGoogle Scholar
  27. Hines J.V., Varani G., Landry S.M., Tinoco I. (1993). J. Am. Chem. Soc. 115:11002–11003CrossRefGoogle Scholar
  28. Hoffman D.W., Holland J.A. (1995). Nucl. Acids. Res. 23:3361–3362CrossRefGoogle Scholar
  29. Hoogstraten C.G., Legault P., Pardi A. (1998). J. Mol. Biol. 284:337–350CrossRefGoogle Scholar
  30. Hoogstraten C.G., Wank J.R., Pardi A. (2000). Biochemistry 39:9951–9958CrossRefGoogle Scholar
  31. Isaacs R.J., Rayens W.S., Spielmann H.P. (2002). J. Mol. Biol. 319:191–207CrossRefGoogle Scholar
  32. Isaacs R.J., Spielmann H.P. (2001). J. Mol. Biol. 307:525–540CrossRefGoogle Scholar
  33. Kay L.E., Torchia D.A., Bax A. (1989). Biochemistry 28:8972–8979CrossRefGoogle Scholar
  34. Ke A., Zhou K., Ding F., Cate J.H., Doudna J.A. (2004). Nature 429:201–205CrossRefADSGoogle Scholar
  35. Kim I., Lukavsky P.J., Puglisi J.D. (2002). J. Am. Chem. Soc. 124:9338–9339CrossRefGoogle Scholar
  36. King G.C., Harper J.W., Xi Z. (1995). Methods. Enzymol. 261:436–450Google Scholar
  37. Kishore A.I., Mayer M.R., Prestegard J.H. (2005). Nucleic Acids Res. 33:e164CrossRefGoogle Scholar
  38. Kline P.C., Serianni A.S. (1990). J. Am. Chem. Soc. 112:7373–7381CrossRefGoogle Scholar
  39. Klooster W.T., Ruble J.R., Craven B.M. (1991). Acta. Crystallogr., B. 47:376–383CrossRefGoogle Scholar
  40. Kojima C., Ono A., Kainosho M., James T.L. (1998). J. Magn. Reson. 135:310–333CrossRefADSGoogle Scholar
  41. Kojima C., Ulyanov N.B., Kainosho M., James T.L. (2001). Biochemistry 40:7239–7246CrossRefGoogle Scholar
  42. Latham M.P., Brown D.J., McCallum S.A., Pardi A. (2005). Chembiochem 6:1492–1505CrossRefGoogle Scholar
  43. Legault P., Hoogstraten C.G., Metlitzky E., Pardi A. (1998). J. Mol. Biol. 284:325–335CrossRefGoogle Scholar
  44. LeMaster D.M., Cronan J.E. Jr. (1982). J. Biol. Chem. 257:1224–1230Google Scholar
  45. LeMaster D.M., Kushlan D.M. (1996). J. Am. Chem. Soc. 118:9255–9264CrossRefGoogle Scholar
  46. LoBrutto R., Bandarian V., Magnusson O.T., Chen X.Y., Schramm V.L., Reed G.H. (2001). Biochemistry 40:9–14CrossRefGoogle Scholar
  47. Lukavsky P.J., Kim I., Otto G.A., Puglisi J.D. (2003). Nat. Struct. Biol. 10:1033–1038CrossRefGoogle Scholar
  48. Nelson D.L., Cox M.M. (2004). Lehninger Principles of Biochemistry. W.H. Freeman, New YorkGoogle Scholar
  49. Nikonowicz E.P., Sirr A., Legault P., Jucker F.M., Baer L.M., Pardi A. (1992). Nucleic Acids Res. 20:4507–4513CrossRefGoogle Scholar
  50. Palmer A.G., Kroenke C.D., Loria J.P. (2001). Methods Enzymol. 339:204–238CrossRefGoogle Scholar
  51. Palmer A.G. III (2004). Chem. Rev. 104:3623–3640CrossRefGoogle Scholar
  52. Parkin D.W., Schramm V.L. (1987). Biochemistry 26:913–920CrossRefGoogle Scholar
  53. Perez-Canadillas J.M., Varani G. (2001). Curr. Opin. Struct. Biol. 11:53–58CrossRefGoogle Scholar
  54. Pley H.W., Flaherty K.M., McKay D.B. (1994). Nature 372:68–74CrossRefADSGoogle Scholar
  55. Rossi P., Harbison G.S. (2001). J. Magn. Reson. 151:1–8CrossRefADSGoogle Scholar
  56. SantaLucia J., Shen L.X., Cai Z.P., Lewis H., Tinoco I. (1995). Nucleic Acids Res. 23:4913–4921CrossRefGoogle Scholar
  57. Shajani Z., Varani G. (2005). J. Mol. Biol. 349:699–715CrossRefGoogle Scholar
  58. Spielmann H.P. (1998). Biochemistry 37:16863–16876CrossRefGoogle Scholar
  59. Szyperski T., Luginbühl P., Otting G., Güntert P., Wüthrich K. (1993). J. Biomolec. NMR 3:151–164Google Scholar
  60. Treiber D.K., Williamson J.R. (2001). Curr. Opin. Struct. Biol. 11:309–314CrossRefGoogle Scholar
  61. Vallurupalli P., Kay L.E. (2005). J. Am. Chem. Soc. 127:6893–6901CrossRefGoogle Scholar
  62. Wagner G., Wüthrich K. (1986). Methods Enzymol. 131:307–326Google Scholar
  63. Williamson J.R., Boxer S.G. (1989). Biochemistry 28:2819–2831CrossRefGoogle Scholar
  64. Yamazaki T., Muhandiram D.R., Kay L.E. (1994). J. Am. Chem. Soc. 116:8266–8278CrossRefGoogle Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  • James E. JohnsonJr
    • 1
  • Kristine R. Julien
    • 1
  • Charles G. Hoogstraten
    • 1
  1. 1.Department of Biochemistry & Molecular BiologyMichigan State UniversityEast LansingUSA

Personalised recommendations