Two-step interrogation then recognition of DNA binding site by Integration Host Factor: an architectural DNA-bending protein.

The dynamics and mechanism of how site-specific DNA-bending proteins initially interrogate potential binding sites prior to recognition have remained elusive for most systems. Here we present these dynamics for Integration Host factor (IHF), a nucleoid-associated architectural protein, using a µs-resolved T-jump approach. Our studies show two distinct DNA-bending steps during site recognition by IHF. While the faster (∼100 µs) step is unaffected by changes in DNA or protein sequence that alter affinity by >100-fold, the slower (1-10 ms) step is accelerated ∼5-fold when mismatches are introduced at DNA sites that are sharply kinked in the specific complex. The amplitudes of the fast phase increase when the specific complex is destabilized and decrease with increasing [salt], which increases specificity. Taken together, these results indicate that the fast phase is non-specific DNA bending while the slow phase, which responds only to changes in DNA flexibility at the kink sites, is specific DNA kinking during site recognition. Notably, the timescales for the fast phase overlap with one-dimensional diffusion times measured for several proteins on DNA, suggesting that these dynamics reflect partial DNA bending during interrogation of potential binding sites by IHF as it scans DNA.

Static Kinks or Flexible Hinges: Multiple Conformations of Bent DNA Bound to Integration Host Factor Revealed by Fluorescence Lifetime Measurements.

Gene regulation depends on proteins that bind to specific DNA sites. Such specific recognition often involves severe DNA deformations, including sharp kinks. It has been unclear how rigid or flexible these protein-induced kinks are. Here, we investigated the dynamic nature of DNA in complex with integration host factor (IHF), a nucleoid-associated architectural protein known to bend one of its cognate sites (35 base pair H') into a U-turn by kinking DNA at two sites. We utilized fluorescence-lifetime-based FRET spectroscopy to assess the distribution of bent conformations in various IHF-DNA complexes. Our results reveal a surprisingly dynamic specific complex: while 78% of the IHF-H' population exhibited FRET efficiency consistent with the crystal structure, 22% exhibited FRET efficiency indicative of unbent or partially bent DNA. This conformational flexibility is modulated by sequence variations in the cognate site. In another site (H1) that lacks the A-tract of H' found on one side of the binding site, the extent of bending in the fully U-bent conformation decreased, and the population in that state decreased to 32%. A similar decrease in the U-bent population was observed with a single base mutation in H' in a consensus region on the other side. Taken together, these results provide important insights into the finely tuned interactions between IHF and its cognate sites that keep the DNA bent (or not) and yield quantitative data on the dynamic equilibrium between different DNA conformations (kinked or not kinked) that depend sensitively on DNA sequence and deformability. Notably, the difference in dynamics between IHF-H' and IHF-H1 reflects the different roles of these complexes in their natural context, in the phage lambda "intasome" (the complex that integrates phage lambda into the E. coli chromosome).