Simultaneous binding and bending of promoter DNA by the TATA binding protein: real time kinetic measurements.

The binding and bending of tetramethylrhodamine-5'-(GGGCTATAAAAGGG) duplex-3'-fluorescein by native Saccharomyces cerevisiae TATA binding protein (TBP) have been investigated using fluorescence resonance energy transfer. Probability distributions derived from fluorescein emission lifetime measurements show a decrease in the mean 3'-fluorescein-5'- rhodamine distance from 56.5 to 46.8 A upon binding of the oligomer to TBP, consistent with the DNA bend observed by X-ray crystallography. The kinetics, monitored in real time using stopped flow fluorimetry, demonstrate simultaneous binding and bending of a TATA box by TBP with a single second-order rate constant of (2.4 +/- 0.3) x 10(6) M-1 s-1 at 30 degrees C.