Dynamics of DNA-Bending in Binding Site Recognition by IHF

Abstract

We present recent progress in monitoring the DNA bending dynamics in site-specific recognition by IHF, an architectural protein from E. coli that recognizes several sites on phage λ DNA, primarily by indirect readout. IHF bends the DNA at its cognate site by nearly 180° over ∼35 bp, creating two kinks in DNA stabilized by intercalation of conserved proline residues located on two β-ribbon arms that wrap around the DNA.Previous stopped-flow and laser T-jump measurements on IHF binding to its cognate H’site revealed that DNA bending in the complex occurs on ∼1-10 ms, similar to the time-scales for thermal disruption of a single base pair in B-DNA. Here we find that inserted mismatches that increase the DNA flexibility at the site of the kinks accelerate the bending rates by nearly the same factor as the corresponding increase in binding affinity. On the other hand, modifications in DNA away from the site of the kink, as well as mutations in IHF, designed to perturb specific protein-DNA contacts, leave the bending rates unchanged despite a ∼60-100-fold decrease in the binding affinity. These results support our earlier conclusion that in the transition state ensemble separating the nonspecific from the specific complex the DNA is bent/kinked, but protein-DNA interactions that stabilize the complex have not yet been made.Our measurements also reveal a rapid (∼100 microseconds) phase in the bending kinetics. In contrast to the relaxation rates for the slow phase, which are affected by modifications in the DNA at the site of the kinks, the relaxation rates for the fast phase appear to be unaffected. This rapid phase may correspond to the wrapping/unwrapping of the β-arms of the protein in a nonspecific binding mode, as IHF scans potential binding sites on genomic DNA.