title: New insights into the transition pathway from nonspecific to specific complex of DNA with Escherichia coli integration host factor.
abstract: To elucidate the nature of the transition-state ensemble along the reaction pathway from a nonspecific protein-DNA complex to the specific complex, we have carried out measurements of DNA bending/unbending dynamics on a cognate DNA substrate in complex with integration host factor (IHF), an architectural protein from E. coli that bends its cognate site by approximately 180 degrees . We use a laser temperature jump to perturb the IHF-DNA complex and monitor the relaxation kinetics with time-resolved FRET measurements on DNA substrates end-labeled with a FRET pair. Previously, we showed that spontaneous bending/kinking of DNA, from thermal disruption of base-pairing/-stacking interactions, may be the rate-limiting step in the formation of the specific complex (Kuznetsov, S. V.; Sugimura, S.; Vivas, P.; Crothers, D. M.; Ansari, A. Proc. Natl. Acad. Sci. USA 2006, 103, 18515). Here, we probe the effect of varying [KCl], which affects the stability of the complex, on this rate-limiting step. We find that below approximately 250 mM KCl, the observed relaxation kinetics are from the unimolecular bending/unbending of DNA, and the relaxation rate kr is independent of [KCl]. Above approximately 300 mM KCl, dissociation of the IHF-DNA complex becomes significant, and the observed relaxation process includes contributions from the association/dissociation step, with kr decreasing with increasing [KCl]. The DNA bending step occurs with a positive activation enthalpy, despite the large negative enthalpy change reported for the specific IHF-DNA complex (Holbrook, J. A.; Tsodikov, O. V.; Saecker, R. M.; Record, M. T., Jr. J. Mol. Biol. 2001, 310, 379). Our conclusion from these studies is that in the uphill climb to the transition state, the DNA is kinked, but with no release of ions, as indicated by the salt-independent behavior of k(r) at low [KCl]. Any release of ions in the unimolecular process, together with conformational changes in the protein-DNA complex that facilitate favorable interactions and that contribute to the negative enthalpy change, must occur as the system leaves the transition state, downhill to the final complex.
authors.affiliation: Department of Physics (M/C 273), and Department of Bioengineering (M/C 063), University of Illinois at Chicago, 845 West Taylor Street, Chicago, Illinois 60607, USA.
authors.forename: Paula
authors.initials: P
authors.lastname: Vivas
authors.forename: Serguei V
authors.initials: SV
authors.lastname: Kuznetsov
authors.forename: Anjum
authors.initials: A
authors.lastname: Ansari
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D034982:Integration Host Factors / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D012680:Sensitivity and Specificity; D012965:Sodium Chloride; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D034982:Integration Host Factors; C440502:integration host factor, E coli; D012965:Sodium Chloride; D004247:DNA