The Role of DNA “Bendability” in the Indirect Read-Out Mechanism of Protein-DNA Interactions

Abstract

Integration host factor (IHF) from E. coli is a DNA-bending protein that recognizes and binds to its specific sites primarily by the indirect read-out mechanism, in which sequence-dependent DNA dynamics and flexibility play an important role. The crystal structure of IHF bound to a 35-bp long cognate site H' indicates that the DNA is kinked at two sites separated by ∼9 bp, resulting in a “U-turn” bend of the DNA. We use laser temperature-jump to perturb the IHF-DNA complex, and time-resolved FRET on end-labeled DNA substrates to monitor the bending/unbending dynamics. In our previous studies, we suggested that spontaneous DNA bending from transient disruption of base-pairing and/or stacking interactions at the site of the kinks may be the rate-limiting step in the transition from the nonspecific to the specific complex. Here, we investigate DNA bending kinetics for substrates with mismatched pairs introduced at the site of the kinks. These internal “loops” are expected to decrease the energetic cost of bending the DNA, which is reflected in the >10-fold increase in the binding affinity. Kinetics measurements on IHF bound to such DNA reveal deviations from single exponential relaxation, indicating two distinct phases. Of particular interest is the observation that the rapid phase has a rate that is 10-20 times faster than the bending rate observed in the IHF-H' complex. Thus, reducing the energetic cost of bending/kinking DNA speeds up the bending rate by nearly the same factor as the increase in binding affinity, indicating that the free energy of the transition state is lowered by the same amount as the free energy of the complex. These results support our earlier conclusion, that spontaneous bending of DNA is the first step in the recognition mechanism.