Stepwise binding and bending of DNA by Escherichia coli integration host factor.
Integration host factor (IHF) is a prokaryotic protein required for the integration of lambda phage DNA into its host genome. An x-ray crystal structure of the complex shows that IHF binds to the minor groove of DNA and bends the double helix by 160 degrees [Rice PA, Yang S, Mizuuchi K, Nash HA (1996) Cell 87:1295-1306]. We sought to dissect the complex formation process into its component binding and bending reaction steps, using stopped-flow fluorimetry to observe changes in resonance energy transfer between DNA-bound dyes, which in turn reflect distance changes upon bending. Different DNA substrates that are likely to increase or decrease the DNA bending rate were studied, including one with a nick in a critical kink position, and a substrate with longer DNA ends to increase hydrodynamic friction during bending. Kinetic experiments were carried out under pseudofirst-order conditions, in which the protein concentration is in substantial excess over DNA. At lower concentrations, the reaction rate rises linearly with protein concentration, implying rate limitation by the bimolecular reaction step. At high concentrations the rate reaches a plateau value, which strongly depends on temperature and the nature of the DNA substrate. We ascribe this reaction limit to the DNA bending rate and propose that complex formation is sequential at high concentration: IHF binds rapidly to DNA, followed by slower DNA bending. Our observations on the bending step kinetics are in agreement with results using the temperature-jump kinetic method.
Sugimura
Sawako
S
Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520, USA.
Crothers
Donald M
DM
D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000138:chemical synthesis / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism / Q000502:physiology*; D034982:Integration Host Factors / Q000378:metabolism / Q000502:physiology*; D009692:Nucleic Acid Heteroduplexes / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D013696:Temperature
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D034982:Integration Host Factors; D009692:Nucleic Acid Heteroduplexes; C440502:integration host factor, E coli