Advertisement

Single-Molecule FRET Characterization of RNA Remodeling Induced by an Antitermination Protein

  • Soraya Ait-Bara
  • Caroline Clerté
  • Emmanuel MargeatEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

Abstract

Single-molecule Förster Resonance Energy Transfer (smFRET) is a useful technique to probe conformational changes within bio-macromolecules. Here, we introduce how to perform smFRET measurements in solution to investigate RNA remodeling and RNA–protein interactions. In particular, we focus on how the close-to-open transition of an antiterminator hairpin is influenced by the binding of the antitermination protein and the competition by oligonucleotides.

Key words

smFRET ALEX Antitermination Hairpin Terminator Transcription Single molecules RNA 

Notes

Acknowledgments

This work was supported by the Agence Nationale de la Recherche (ANR 2010 BLAN 1525 01 to E.M.), a “Chercheur d’Avenir” grant from the Region Languedoc Roussillon to E.M., the GIS “IBiSA: Infrastructures en Biologie Sante et Agronomie” and a postdoctoral grant from the Université Montpellier I to S.A.-B.

References

  1. 1.
    Weiss S (2000) Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy. Nat Struct Biol 7:724–729PubMedCrossRefGoogle Scholar
  2. 2.
    Felekyan S, Sanabria H, Kalinin S et al (2013) Analyzing Forster resonance energy transfer with fluctuation algorithms. Methods Enzymol 519:39–85PubMedCrossRefGoogle Scholar
  3. 3.
    Clerte C, Declerck N, Margeat E (2013) Competitive folding of anti-terminator/terminator hairpins monitored by single molecule FRET. Nucleic Acids Res 41:2632–2643PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    van Tilbeurgh H, Declerck N (2001) Structural insights into the regulation of bacterial signalling proteins containing PRDs. Curr Opin Struct Biol 11:685–693PubMedCrossRefGoogle Scholar
  5. 5.
    Aymerich S, Steinmetz M (1992) Specificity determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BglG/SacY family. Proc Natl Acad Sci U S A 89:10410–10414PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Schnetz K, Stulke J, Gertz S et al (1996) LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family. J Bacteriol 178:1971–1979PubMedCentralPubMedGoogle Scholar
  7. 7.
    Johnson IaS MTZ (2010) Molecular probes handbook. A guide to fluorescent probes and labeling technologies, 11th edGoogle Scholar
  8. 8.
    Van Tilbeurgh H, Le Coq D, Declerck N (2001) Crystal structure of an activated form of the PTS regulation domain from the LicT transcriptional antiterminator. EMBO J 20:3789–3799PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Declerck N, Vincent F, Hoh F et al (1999) RNA recognition by transcriptional antiterminators of the BglG/SacY family: functional and structural comparison of the CAT domain from SacY and LicT. J Mol Biol 294:389–402PubMedCrossRefGoogle Scholar
  10. 10.
    Kapanidis AN, Lee NK, Laurence TA et al (2004) Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc Natl Acad Sci U S A 101:8936–8941PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Kapanidis AN, Laurence TA, Lee NK et al (2005) Alternating-laser excitation of single molecules. Acc Chem Res 38:523–533PubMedCrossRefGoogle Scholar
  12. 12.
    Kapanidis AN, Heilemann M, Margeat E, Kong X, Nir E, Weiss S (2008) Alternating-laser excitation of single molecules. In: Selvin PR, Ha T (eds) Single-molecule techniques: a laboratory manual. CSHL Press, New York, pp 85–119Google Scholar
  13. 13.
    Proudnikov D, Mirzabekov A (1996) Chemical methods of DNA and RNA fluorescent labeling. Nucleic Acids Res 24:4535–4542PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Persson T, Willkomm DK, Hartmann RK (2005) T4 RNA ligase. In: Bindereif A, Schön A, Wethof E, Hartmann RK (eds) Handbook of RNA biochemistry. Wiley, Weinheim, pp 53–74CrossRefGoogle Scholar
  15. 15.
    Ha T (2001) Single-molecule fluorescence resonance energy transfer. Methods 25:78–86PubMedCrossRefGoogle Scholar
  16. 16.
    Clegg RM (1992) Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol 211:353–388PubMedCrossRefGoogle Scholar
  17. 17.
    Holden SJ, Uphoff S, Hohlbein J et al (2010) Defining the limits of single-molecule FRET resolution in TIRF microscopy. Biophys J 99:3102–3111PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Eggeling CBS, Brand L, Fries JR, Schaffer J, Volkmer A, Seidel CA (2001) Data registration and selective single-molecule analysis using multi-parameter fluorescence detection. J Biotechnol 13:163–180CrossRefGoogle Scholar
  19. 19.
    Lee NK, Kapanidis AN, Wang Y, Michalet X, Mukhopadhyay J, Ebright RH, Weiss S (2005) Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. Biophys J 88:2939–2943PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Hendrix J, Lamb DC (2013) Pulsed interleaved excitation: principles and applications. Methods Enzymol 518:205–243PubMedCrossRefGoogle Scholar
  21. 21.
    Olofsson L, Margeat E (2013) Pulsed interleaved excitation fluorescence spectroscopy with a supercontinuum source. Opt Express 21:3370–3378PubMedCrossRefGoogle Scholar
  22. 22.
    Nir E, Michalet X, Hamadani KM et al (2006) Shot-noise limited single-molecule FRET histograms: comparison between theory and experiments. J Phys Chem B 110:22103–22124PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5:507–516PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Vogelsang J, Kasper R, Steinhauer C et al (2008) A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew Chem Int Ed Engl 47:5465–5469PubMedCrossRefGoogle Scholar
  25. 25.
    Ha T, Tinnefeld P (2012) Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. Annu Rev Phys Chem 63:595–617PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Yang Y, Declerck N, Manival X et al (2002) Solution structure of the LicT-RNA antitermination complex: CAT clamping RAT. EMBO J 21:1987–1997PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Soraya Ait-Bara
    • 1
    • 2
    • 3
  • Caroline Clerté
    • 1
    • 2
    • 3
  • Emmanuel Margeat
    • 1
    • 2
    • 3
    Email author
  1. 1.CNRS UMR5048Centre de Biochimie StructuraleMontpellierFrance
  2. 2.INSERM U1054MontpellierFrance
  3. 3.Universités Montpellier I et IIMontpellierFrance

Personalised recommendations