Single-molecule evidence for a chemical ratchet in binding between the cam repressor and its operator

Abstract

The affinity for regulator–operator binding on DNA sometimes depends on the length of the DNA harboring the operator, which is known as the antenna effect. One-dimensional diffusion along DNA has been suggested to be the cause, but this may contradict the binding affinity independent of the reaction pathways, which is derived from the detailed balance of the reaction at equilibrium. Recently, the chemical ratchet was proposed to solve this contradiction by suggesting a stationary state containing microscopic non-equilibrium. In a single-molecule observation, P. putida CamR molecules associate with their operator via one-dimensional diffusion along the DNA, while they mostly dissociated from the operator without the diffusion. Consistently, the observed overall association rate was dependent on the DNA length, while the overall dissociation rate was not, leading to an antenna effect. E. coli RNA polymerase did not show this behavior, and thus it is a specific property of a protein. The bipartite interaction domains containing the helix-turn-helix motif are speculated to be one of the possible causes. The biological significance of the chemical ratchet and a model for its microscopic mechanism are also discussed.