Limits...
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.

Esadze A, Kemme CA, Kolomeisky AB, Iwahara J - Nucleic Acids Res. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

Related in: MedlinePlus

Ionic-strength dependence of sliding. (A) Length dependence of ka constants at 40, 60, 110 and 150 mM. The horizontal axis represents DNA length in bp. Solid lines are the best-fit curves obtained using Equations (1)–(4). The parameter L, which represents the number of binding sites on the probe DNA, was set to (DNA length in bp) −8 (see the main text). The one-dimensional diffusion coefficient D1 and the effective sliding length λ were optimized in the fitting calculations. Dtot = 2.5 nM and Ctot = 2 μM were used in the measurements. (B) The one-dimensional diffusion coefficient D1 for sliding of the Egr-1 zinc-finger protein as a function of KCl concentration. (C) The effective sliding length λ as a function of ionic strength.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4066804&req=5

Figure 2: Ionic-strength dependence of sliding. (A) Length dependence of ka constants at 40, 60, 110 and 150 mM. The horizontal axis represents DNA length in bp. Solid lines are the best-fit curves obtained using Equations (1)–(4). The parameter L, which represents the number of binding sites on the probe DNA, was set to (DNA length in bp) −8 (see the main text). The one-dimensional diffusion coefficient D1 and the effective sliding length λ were optimized in the fitting calculations. Dtot = 2.5 nM and Ctot = 2 μM were used in the measurements. (B) The one-dimensional diffusion coefficient D1 for sliding of the Egr-1 zinc-finger protein as a function of KCl concentration. (C) The effective sliding length λ as a function of ionic strength.

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.


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.

Esadze A, Kemme CA, Kolomeisky AB, Iwahara J - Nucleic Acids Res. (2014)

Ionic-strength dependence of sliding. (A) Length dependence of ka constants at 40, 60, 110 and 150 mM. The horizontal axis represents DNA length in bp. Solid lines are the best-fit curves obtained using Equations (1)–(4). The parameter L, which represents the number of binding sites on the probe DNA, was set to (DNA length in bp) −8 (see the main text). The one-dimensional diffusion coefficient D1 and the effective sliding length λ were optimized in the fitting calculations. Dtot = 2.5 nM and Ctot = 2 μM were used in the measurements. (B) The one-dimensional diffusion coefficient D1 for sliding of the Egr-1 zinc-finger protein as a function of KCl concentration. (C) The effective sliding length λ as a function of ionic strength.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4066804&req=5

Figure 2: Ionic-strength dependence of sliding. (A) Length dependence of ka constants at 40, 60, 110 and 150 mM. The horizontal axis represents DNA length in bp. Solid lines are the best-fit curves obtained using Equations (1)–(4). The parameter L, which represents the number of binding sites on the probe DNA, was set to (DNA length in bp) −8 (see the main text). The one-dimensional diffusion coefficient D1 and the effective sliding length λ were optimized in the fitting calculations. Dtot = 2.5 nM and Ctot = 2 μM were used in the measurements. (B) The one-dimensional diffusion coefficient D1 for sliding of the Egr-1 zinc-finger protein as a function of KCl concentration. (C) The effective sliding length λ as a function of ionic strength.
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.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus