title: Mapping the transition state for DNA bending by IHF.
abstract: How DNA-bending proteins recognize their specific sites on DNA remains elusive, particularly for proteins that use indirect readout, which relies on sequence-dependent variations in DNA flexibility/bendability. The question remains as to whether the protein bends the DNA (protein-induced bending) or, alternatively, "prebent" DNA conformations are thermally accessible, which the protein captures to form the specific complex (conformational capture). To distinguish between these mechanisms requires characterization of reaction intermediates and, in particular, snapshots of the transition state along the recognition pathway. We present such a snapshot, from measurements of DNA bending dynamics in complex with Escherichia coli integration host factor (IHF), an architectural protein that bends specific sites on λ-DNA in a U-turn by creating two sharp kinks in DNA. Fluorescence resonance energy transfer measurements in response to laser temperature-jump perturbation monitor DNA bending. We find that nicks or mismatches that enhance DNA flexibility at the site of the kinks show 3- to 4-fold increase in DNA bending rates that reflect a 4- to 11-fold increase in binding affinities, while sequence modifications away from the kink sites, as well as mutations in IHF designed to destabilize the complex, have negligible effect on DNA bending rates despite >250-fold decrease in binding affinities. These results support the scenario that the bottleneck in the recognition step for IHF is spontaneous kinking of cognate DNA to adopt a partially prebent conformation and point to conformational capture as the underlying mechanism of initial recognition, with additional protein-induced bending occurring after the transition state.
authors.affiliation: Department of Physics (M/C 273), University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607, USA.
authors.forename: Paula
authors.initials: P
authors.lastname: Vivas
authors.forename: Yogambigai
authors.initials: Y
authors.lastname: Velmurugu
authors.forename: Serguei V
authors.initials: SV
authors.lastname: Kuznetsov
authors.forename: Phoebe A
authors.initials: PA
authors.lastname: Rice
authors.forename: Anjum
authors.initials: A
authors.lastname: Ansari
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D034982:Integration Host Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation
chemical_list: D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D004247:DNA