Advertisement

Using FRET to Monitor Protein-Induced DNA Bending: The TBP-TATA Complex as a Model System

  • Rebecca H. Blair
  • James A. Goodrich
  • Jennifer F. Kugel
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 977)

Abstract

Proteins that bind to DNA can elicit changes in DNA conformation, such as bending and looping, which are important signals for later events such as transcription. TATA-binding protein (TBP) is one example of a protein that elicits a conformational change in DNA; TBP binds and sharply bends its recognition sequence, which is thought to facilitate the recruitment of other protein factors. Here we describe the use of fluorescence resonance energy transfer (FRET) to evaluate DNA bending using TBP as a model system. FRET is a useful technique to measure changes in DNA conformation due to protein binding because small changes in the distance between two fluorophores (2–10 nm) translate into large changes in energy transfer.

Key words

FRET DNA bending Protein–DNA interaction TBP Fluorophore 

Notes

Acknowledgments

This work was supported by grant MCB-0919935 from the National Science Foundation.

References

  1. 1.
    Starr DB, Hawley DK (1991) TFIID binds the minor groove of the TATA box. Cell 67: 1231–1240PubMedCrossRefGoogle Scholar
  2. 2.
    Starr DB, Hoopes BC, Hawley DK (1995) DNA bending is an important component of site-specific recognition by the TATA binding protein. J Mol Biol 250:434–446PubMedCrossRefGoogle Scholar
  3. 3.
    Kim JL, Nikolov DB, Burley SK (1993) Co-crystal structure of TBP recognizing the minor groove of a TATA element. Nature 365:520–527PubMedCrossRefGoogle Scholar
  4. 4.
    Kim Y, Geiger JH, Hahn S et al (1993) Crystal structure of a yeast TBP/TATA-box complex. Nature 365:512–520PubMedCrossRefGoogle Scholar
  5. 5.
    Nikolov DB, Chen H, Halay ED et al (1996) Crystal structure of a human TATA box-binding protein/TATA element complex. Proc Natl Acad Sci USA 93:4862–4867PubMedCrossRefGoogle Scholar
  6. 6.
    Lee DK, Horikoshi M, Roeder RG (1991) Interaction of TFIID in the minor groove of the TATA element. Cell 67:1241–1250PubMedCrossRefGoogle Scholar
  7. 7.
    Wu J, Parkhurst KM, Powell RM et al (2001) DNA bends in TATA-binding protein-TATA complexes in solution are DNA sequence-dependent. J Biol Chem 276:14614–14622PubMedCrossRefGoogle Scholar
  8. 8.
    Whittington JE, Delgadillo RF, Attebury TJ et al (2008) TATA-binding protein recognition and bending of a consensus promoter are ­protein species dependent. Biochemistry 47: 7264–7273PubMedCrossRefGoogle Scholar
  9. 9.
    Hieb AR, Halsey WA, Betterton MD et al (2007) TFIIA changes the conformation of the DNA in TBP/TATA complexes and increases their kinetic stability. J Mol Biol 372:619–632PubMedCrossRefGoogle Scholar
  10. 10.
    Kugel JF (2008) Using FRET to Measure the Angle at Which a Protein Bends DNA. Biochem Mol Biol Edu 36:341–346CrossRefGoogle Scholar
  11. 11.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, New YorkCrossRefGoogle Scholar
  12. 12.
    Johnson I, Spence MTZ (2010) Molecular Probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies, 11th edn. Life Technologies, OregonGoogle Scholar
  13. 13.
    Wu J, Parkhurst KM, Powell RM et al (2001) DNA sequence-dependent differences in TATA-binding protein-induced DNA bending in solution are highly sensitive to osmolytes. J Biol Chem 276:14623–14627PubMedCrossRefGoogle Scholar
  14. 14.
    Weaver JR, Kugel JF, Goodrich JA (2005) The sequence at specific positions in the early transcribed region sets the rate of transcript synthesis by RNA polymerase II in vitro. J Biol Chem 280:39860–39869PubMedCrossRefGoogle Scholar
  15. 15.
    Goodrich JA, Kugel JF (2007) Binding and kinetics for molecular biologists. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Rebecca H. Blair
    • 1
  • James A. Goodrich
    • 1
  • Jennifer F. Kugel
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of ColoradoBoulderUSA

Personalised recommendations