Stepwise binding and bending of DNA by Escherichia coli integration host factor

Abstract

Integration host factor (IHF) is a prokaryotic protein required for the integration of 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°[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 DNAbound 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.kinetics ͉ stopped flow ͉ FRET P rotein-DNA interactions control essential cellular processes, including enzymatic, regulatory, and structural roles. Significant conformational changes in DNA are often induced upon protein binding to DNA, both in specific and nonspecific protein-DNA interactions. In many sequence-specific interactions between protein and DNA, intrinsic DNA distortion and flexibility are key players in recognition of cognate sequences (indirect read-out); other factors include formation of hydrogen bonds and other contact interactions at the local level (direct read-out) (1, 2). Indirect read-out is known to play a major role in DNA sequence specificity in certain systems such as in the complexes of DNA with E2 (3), EcoRV (4), HincII (4), yeast TATA box-binding protein (5), and the integration host factor (IHF) (6, 7). However, the transient mechanisms of protein-DNA association and dissociation reactions remain largely elusive.IHF is a small Escherichia coli heterodimeric protein (Ϸ10 kDa each) that can bend DNA and act as an architectural factor in many cellular activities (8, 9). It is involved in DNA replication and transcriptional regulation and in DNA condensation in processes that require DNA bending so as to bring two distant DNA sequences into proximity (8, 9). Its presence is crucial for phage DNA recombination; it has been shown to bind to its specific binding sequences with 10 3 to 10 4 times higher affinity compared with nonspecific sites (10-12). DNase I and hydroxyl radical foo...

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

“…The temperature-dependence of the relaxation rates for the bending͞unbending step obtained from the T-jump measurements are well described by an Arrhenius equation, with an apparent activation energy of Ϸ14.5 kcal͞mol, which is consistent with the activation energy of Ϸ16 Ϯ 3 kcal͞mol obtained from the stopped-flow data (22). A global fit to the relaxation rates from the T-jump and stopped-flow data combined yields an apparent activation energy of Ϸ18 Ϯ 3 kcal͞mol.…”

“…It should be kept in mind that changes in the far-UV CD spectra monitor only the secondary structure disruption and are not proof that the tertiary structure is not disrupted. Nevertheless, the CD experiments, together with the consistent set of relaxation rates for the unimolecular process obtained from the stopped-flow measurements (22), and T-jump measurements, as described in the next section, suggest that IHF is in its native conformation up to at least 40°C.…”

“…Stopped-flow measurements were carried out to monitor the overall association rate for the binding of IHF to HЈ, under pseudofirst order conditions, with IHF concentration in large excess over the DNA oligomer concentration (22). The relaxation kinetics, obtained with a time resolution of Ϸ3 ms, were monitored by timeresolved changes in the fluorescence emission of fluorescein, and are well described by a single exponential.…”

“…A motivation for investigating the bending dynamics of HЈ bound to IHF comes from the results of Sugimura and Crothers (22), who show in the accompanying article that the observed relaxation rate for IHF binding to HЈ, obtained by using stoppedflow with time-resolved FRET, saturates at high concentrations of IHF. These results suggest that the binding of IHF to HЈ and the bending of the HЈ substrate occur sequentially, and that at high IHF concentrations the bending of DNA becomes the rate-limiting step.…”

Basic statistical recipes for the emergence of biochemical discernment

“…This mechanism allows reaching precise molecular stereo-adjustments between weakly preorganized but supple macromolecules. The induced fit mechanism has been invoked for enzymes with lid-gated sites (Sullivan and Holyoak, 2008) or for DNAbending proteins such as IHF (Khrapunov et al, 2006;Sugimura and Crothers, 2006). In the two-step mechanism proposed in these studies, IHF binds first to linear DNA, which then progressively bends around IHF.…”