Browsing by Author "Iwahara, Junji"
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Item Discrete-state stochastic kinetic models for target DNA search by proteins: Theory and experimental applications(Elsevier, 2021) Iwahara, Junji; Kolomeisky, Anatoly B.; Center for Theoretical Biological PhysicsTo perform their functions, transcription factors and DNA-repair/modifying enzymes randomly search DNA in order to locate their specific targets on DNA. Discrete-state stochastic kinetic models have been developed to explain how the efficiency of the search process is influenced by the molecular properties of proteins and DNA as well as by other factors such as molecular crowding. These theoretical models not only offer explanations on the relation of microscopic processes to macroscopic behavior of proteins, but also facilitate the analysis and interpretation of experimental data. In this review article, we provide an overview on discrete-state stochastic kinetic models and explain how these models can be applied to experimental investigations using stopped-flow, single-molecule, nuclear magnetic resonance (NMR), and other biophysical and biochemical methods.Item Positive and negative impacts of nonspecific sites during target location by a sequence-specific DNA-binding protein: origin of the optimal search at physiological ionic strength(Oxford University Press, 2014) Esadze, Alexandre; Kemme, Catherine A.; Kolomeisky, Anatoly B.; Iwahara, Junji; Center for Theoretical Biological PhysicsThe inducible transcription factor Egr-1, which recognizes a 9-bp target DNA sequence via three zinc-finger domains, rapidly activates particular genes upon cellular stimuli such as neuronal signals and vascular stresses. Here, using the stopped-flow fluorescence method, we measured the target search kinetics of the Egr-1 zinc-finger protein at various ionic strengths between 40 and 400 mM KCl and found the most efficient search at 150 mM KCl. We further investigated the kinetics of intersegment transfer, dissociation, and sliding of this protein on DNA at distinct concentrations of KCl. Our data suggest that Egr-1's kinetic properties are well suited for efficient scanning of chromosomal DNA in vivo. Based on a newly developed theory, we analyzed the origin of the optimal search efficiency at physiological ionic strength. Target association is accelerated by nonspecific binding to nearby sites and subsequent sliding to the target as well as by intersegment transfer. Although these effects are stronger at lower ionic strengths, such conditions also favor trapping of the protein at distant nonspecific sites, decelerating the target association. Our data demonstrate that Egr-1 achieves the optimal search at physiological ionic strength through a compromise between the positive and negative impacts of nonspecific interactions with DNA.