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...

Regulation of gene expression involves formation of specific protein-DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNAbinding sites. Previous kinetics measurements on several DNAbending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending͞unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from Ϸ2 ms ؊1 at Ϸ37°C to Ϸ40 ms ؊1 at Ϸ10°C and correspond to an activation energy of Ϸ14 ؎ 3 kcal͞mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend͞kink DNA before forming a tight complex with IHF.DNA bending kinetics ͉ laser temperature jump ͉ protein-DNA interactions ͉ time-resolved FRET measurements

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F-ATPase is a rotary motor enzyme with the biological function of synthesizing ATP, the ''fuel molecule'' of cells. A subsystem of F-ATPase, F1-ATPase, functions in reverse: it hydrolyses ATP into ADP and inorganic phosphate. During this process, the rotation of F1-ATPase's shaft-like rotor is driven by chemical free energy. Samples of paracoccus denitrificans F1-ATPase are studied in single-molecule imaging experiments while the enzyme undergoes ATP synthesis. This single-molecule experiment is performed through the use of a 40 nanometer gold nano-probe used to record rotation angles at a microsecond temporal resolution. The biomotor enzyme experiences rotary motion during the hydrolysis of ATP, and periodically transitions between various states (angular dwells) in a step-like manner. The substeps observed in other species of F1-ATPase appear to be absent in the paracoccus denitrificans species, so a goal of our work is to use a previous multi-state theory for the angle-dependent velocity to detect any ''hidden'' substeps with too short a lifespan to be seen in the experimental trajectories. Additionally, the Brownian motion of the probe prohibits analysis of the possible state configuration of F1-ATPase through direct observation. Due to this, we have developed an automated method utilizing the enzyme's rotary progression as well as likelihood theorems to separate dwell and transitional states within single molecule trajectories. The method thereby allows an analysis of the state model of experimental results using calculations based on the existing theory.