  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.
