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.
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Starr DB, Hawley DK (1991) TFIID binds the minor groove of the TATA box. Cell 67: 1231–1240
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 250:434–446
Kim JL, Nikolov DB, Burley SK (1993) Co-crystal structure of TBP recognizing the minor groove of a TATA element. Nature 365:520–527
Kim Y, Geiger JH, Hahn S et al (1993) Crystal structure of a yeast TBP/TATA-box complex. Nature 365:512–520
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 93:4862–4867
Lee DK, Horikoshi M, Roeder RG (1991) Interaction of TFIID in the minor groove of the TATA element. Cell 67:1241–1250
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 276:14614–14622
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: 7264–7273
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 372:619–632
Kugel JF (2008) Using FRET to Measure the Angle at Which a Protein Bends DNA. Biochem Mol Biol Edu 36:341–346
Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, New York
Johnson I, Spence MTZ (2010) Molecular Probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies, 11th edn. Life Technologies, Oregon
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 276:14623–14627
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 280:39860–39869
Goodrich JA, Kugel JF (2007) Binding and kinetics for molecular biologists. Cold Spring Harbor Laboratory Press, New York
Acknowledgments
This work was supported by grant MCB-0919935 from the National Science Foundation.
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Blair, R.H., Goodrich, J.A., Kugel, J.F. (2013). Using FRET to Monitor Protein-Induced DNA Bending: The TBP-TATA Complex as a Model System. In: Bina, M. (eds) Gene Regulation. Methods in Molecular Biology, vol 977. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-284-1_16
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DOI: https://doi.org/10.1007/978-1-62703-284-1_16
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Publisher Name: Humana Press, Totowa, NJ
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