Quantitative transcription factor binding kinetics at the single-molecule level

TL;DR

This study used single-molecule fluorescence microscopy to quantify the binding kinetics of bacteriophage lambda repressor CI to DNA, revealing how association and dissociation rates depend on DNA length, protein concentration, and binding affinity.

Contribution

It provides the first detailed single-molecule analysis of CI-DNA binding kinetics, including direct measurement of rate constants and their dependence on experimental parameters.

Findings

CI binding to operator DNA is independent of flanking non-operator DNA length.

Association and dissociation follow Poisson statistics, allowing rate constant determination.

Binding kinetics vary with DNA length, protein concentration, and affinity.

Abstract

We have investigated the binding interaction between the bacteriophage lambda repressor CI and its target DNA using total internal reflection fluorescence microscopy. Large, step-wise changes in the intensity of the red fluorescent protein fused to CI were observed as it associated and dissociated from individually labeled single molecule DNA targets. The stochastic association and dissociation were characterized by Poisson statistics. Dark and bright intervals were measured for thousands of individual events. The exponential distribution of the intervals allowed direct determination of the association and dissociation rate constants, ka and kd respectively. We resolved in detail how ka and kd varied as a function of 3 control parameters, the DNA length L, the CI dimer concentration, and the binding affinity. Our results show that although interaction with non-operator DNA sequences are observable, CI binding to the operator site is not dependent on the length of flanking non-operator DNA.