SIMULTANEOUS BINDING AND BENDING OF PROMOTER DNA BY THE TATA-BINDING PROTEIN - REAL-TIME KINETIC MEASUREMENTS

The binding and bending of odamine-5'-(GGGCTATAAAAGGG)(duplex)-3'-fluo rescein by native Saccharomyces cerevisiae TATA binding protein (TBP) have been investigated using fluorescence resonance energy transfer. P robability distributions derived from fluorescein emission lifetime me asurements show a decrease in the mean 3'-fluorescein-5'-rhodamine dis tance from 56.5 to 46.8 Angstrom upon binding of the oligomer to TBP, consistent with the DNA bend observed by X-ray crystallography. The ki netics, monitored in real time using stopped flow fluorimetry, demonst rate simultaneous binding and bending of a TATA box by TBP with a sing le second-order rate constant of (2.4 +/- 0.3) x 10(6) M(-1) s(-1) at 30 degrees C.