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Proceedings Paper

Dynamics of TBP binding to the TATA box
Author(s): Peter Schluesche; Gregor Heiss; Michael Meisterernst; Don C. Lamb
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Paper Abstract

Gene expression is highly controlled and regulated in living cells. One of the first steps in gene transcription is recognition of the promoter site by the TATA box Binding Protein (TBP). TBP recruits other transcriptions factors and eventually the RNA polymerase II to transcribe the DNA in mRNA. We developed a single pair Förster Resonance Energy Transfer (spFRET) assay to investigate the mechanism of gene regulation. Here, we apply this assay to investigate the initial binding process of TBP to the adenovirus major late (AdML) promoter site. From the spFRET measurements, we were able to identify two conformations of the TBP-DNA complex that correspond to TBP bound in the correct and the opposite orientation. Increased incubation times or the presence of the transcription factor TFIIA improved the alignment of TBP on the promoter site. Binding of TBP to the TATA box shows a rich dynamics with abrupt transitions between multiple FRET states. A frame-wise histogram analysis revealed the presence of at least six discrete states, showing that TBP binding is more complicated than previously thought. Hence, the spFRET assay is very sensitive to the conformation of the TBP-DNA complex and is very promising tool for investigating the pathway of TBP binding in detail.

Paper Details

Date Published: 21 February 2008
PDF: 8 pages
Proc. SPIE 6862, Single Molecule Spectroscopy and Imaging, 68620E (21 February 2008); doi: 10.1117/12.769177
Show Author Affiliations
Peter Schluesche, Ludwig-Maximilians-Univ. München (Germany)
Gregor Heiss, Ludwig-Maximilians-Univ. München (Germany)
Michael Meisterernst, Helmholtz Ctr. Munich German Research Ctr. for Environmental Health (Germany)
Institute for Cell and Tumor Biology, Westfalian Wilhelms Univ. (Germany)
Don C. Lamb, Ludwig-Maximilians-Univ. München (Germany)
Univ. of Illinois at Urbana-Champaign (United States)

Published in SPIE Proceedings Vol. 6862:
Single Molecule Spectroscopy and Imaging
Jörg Enderlein; Zygmunt K. Gryczynski; Rainer Erdmann, Editor(s)

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