LexA-DNA Bond Strength by Single Molecule Force Spectroscopy

Abstract

The SOS system of Escherichia coli is coordinated by two proteins: LexA, a repressor protein of several unlinked genes, and the coprotease RecA. As known to date LexA controls 31 genes with slightly different DNA binding motifs allowing for a variable degree of repression from one gene to the other. Besides the SOS system LexA plays an important role in the regulation of transcription. The protein regulates transcription by using particular motifs to bind DNA, the helix-turn-helix motif. Here, we employed AFM-based single molecule force spectroscopy to characterize the interaction of LexA protein with two different DNA motifs: recA and yebG. We measured the dissociation rates to be 0.045 s(-1) for recA and 0.13 s(-1) for yebG, respectively, which is in accordance with the predicted higher affinity between LexA-recA compared to LexA-yebG. The widths of the binding potentials were determined to be 5.4 +/- 1 angstroms and 4.9 +/- 0.5 angstroms, respectively. This short-ranged potential is characteristic for a stiff hydrogen-bonding network between protein and DNA. The unbinding occurs in a breakup rather than a gradual sliding.

The Reaction-Diffusion Master Equation as an Asymptotic Approximation of Diffusion to a Small Target

“…This suggests that for physical reaction-radii of one nanometer or less the reaction-diffusion master equation may be a good approximation to a diffusion limited reaction. While physical reaction-radii have not been experientially determined for most biological reactions, it has been found experimentally that the LexA DNA binding protein has a physical binding potential of width ∼ 5Å [30]. We caution, however, that these results are only valid for the truncated perturbation expansions, and do not necessarily hold for the error between the exact solutions p h (x, t) and p(x, t).…”

Force measurements with the atomic force microscope: Technique, interpretation and applications

“…Several experiments also have been performed with DNA strands [1078,[1143][1144][1145][1146], DNA-ligands [1147][1148][1149][1150], RNA [1151], RNA-ligands [1152], peptides [1153], and other complementary molecules [389,1154,1155]. In particular, Schumakovitch et al [1156] have studied the temperature dependence of unbinding forces between complementary DNA strands.…”

Single‐Molecule Experiments in Synthetic Biology: An Approach to the Affinity Ranking of DNA‐Binding Peptides

“…[15] The direct investigation of specific, native protein-DNA interactions has been reported recently. [23][24][25] In our studies reported herein, we applied AFM singlemolecule force spectroscopy to study the specific binding between the peptides listed in Table 1 and the DNA target sequence. Each peptide was covalently immobilized to an amino-functionalized mica surface in a directed manner with the short C-terminal linker 1,8-diamino-3,6-dioxaoctane and the cross-linker BS 3 (bis(sulfosuccinimidyl)suberate; Figure 1 b).…”