Simultaneous Binding and Bending of Promoter DNA by the TATA Binding Protein: Real Time Kinetic Measurements

Abstract

The binding and bending of tetramethylrhodamine−5‘-(GGGC TATAAAAG GG)_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 Å 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) × 10⁶ M^-1 s^-1 at 30 °C.