Regensburg 2007 – wissenschaftliches Programm
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BP: Fachverband Biologische Physik
BP 9: Regulation and Signaling
BP 9.8: Vortrag
Dienstag, 27. März 2007, 12:00–12:15, H43
The Mechanism of Gene Repression Revealed by spFRET Investigations of TBP-NC2 Complexes — •Peter Schluesche1, Gertraud Stelzer2, Elisa Piaia2, Christoph Braeuchle1, Michael Meisterernst2, and Don C. Lamb1 — 1Department for Physical Chemistry, Ludwig-Maximilians-University, Butenandtstr. 11, 81377 München — 2GSF-National Research Center for Environment and Health, Department of Gene Expression, Marchioninistr. 25, 81377 München
The initiation of DNA transcription starts with the binding of the TATA-Box Binding Protein (TBP) to the gene promoter site on the DNA. The transcription can be inhibited by the regulatory protein Negative Cofactor 2 (NC2) which forms a complex with the DNA bound TBP and represses gene expression. Recent results of biochemical experiments suggest that the TBP becomes mobile along the DNA upon the binding of NC2. Thus, transcription inhibition by NC2 could be explained by the dislocation of TBP from the promoter site rather than by steric hindrances as currently thought. To test this hypothesis, we have performed fluorescence resonance energy transfer (FRET) experiments on single TBP-NC2 complexes bound to DNA using Total Internal Reflection Fluorescence Microscopy with dual color detection. TBP was labeled specifically with a FRET-donor molecule and a TATA sequence containing dsDNA was labeled with a FRET-acceptor. By observing fluctuations in the FRET efficiency of individual complexes upon NC2 binding, we confirmed the mobility of the TBP-NC2 complexes along the DNA.