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.
Bottom Line: 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.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.
Affiliation: Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA.Show MeSH
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Mentions: Target search kinetics of the Egr-1 zinc-finger protein as a function of ionic strength. (A) Stopped-flow fluorescence assay of the target search kinetics. The fluorescence time-course data shown were obtained using 113-bp probe DNA (Dtot = 2.5 nM), nonspecific 28-bp DNA (Ctot = 2000 nM) and protein (Ptot = 50 nM) at 60, 150 and 300 mM KCl. Concentrations Ptot and Ctot were varied in other measurements. (B) Apparent second-order rate constants ka for target association of the Egr-1 zinc-finger protein in the presence of 2000 nM nonspecific 28-bp DNA. The experiment was performed using 113-bp probe DNA at 40, 60, 80, 110, 150, 190, 230, 300 and 400 mM KCl. Values of ka constant were determined from the pseudo-first-order rate constants for target association at different concentrations of the protein. The solid line shown in the plot is a best-fit curve obtained using Equations (1)–(5) and the counterion condensation theory for the kinetic and thermodynamic parameters (see the main text). The data are shown on a logarithmic scale for each axis. For this panel and for Figures 2 and 3, the error bars represent the standard error of the mean (SEM) estimated from 6–10 replicates. For data points with no error bars, the SEMs were smaller than the size of the symbols.
Affiliation: Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA.