One-dimensional diffusion of TrpR along DNA enhances its affinity for the operator by chemical ratchet mechanism

Abstract

Several DNA-binding proteins show the affinities for their specific DNA sites that positively depend on the length of DNA harboring the sites, i. e. antenna effect. DNA looping can cause the effect for proteins with two or more DNA binding sites, i. e. the looping mechanism. One-dimensional diffusion also has been suggested to cause the effect for proteins with single DNA sites, the diffusion mechanism, which could violate detailed balance. We addressed which mechanism is possible for E. coli TrpR showing 104-fold antenna effect with a single DNA binding site. When a trpO-harboring DNA fragment was connected to a nonspecific DNA with biotin-avidin connection, the otherwise sevenfold antenna effect disappeared. This result denies the looping mechanism with an unknown second DNA binding site. The 3.5-fold repression by TrpR in vivo disappeared when a tight LexA binding site was introduced at various sites near the trpO, suggesting that the binding of LexA blocks one-dimensional diffusion causing the antenna effect. These results are consistent with the chemical ratchet recently proposed for TrpR-trpO binding to solve the deviation from detailed balance, and evidence that the antenna effect due to one-dimensional diffusion exists in cells.

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

“…In the study of TrpR– trpO binding, 8 a binding site of LexA was introduced at a distal position where direct physical contact between LexA and TrpR was not possible. Notably, the binding of cellular LexA to the artificial site suppressed the repression by TrpR in vivo , and this was overcome by the over-production of TrpR, suggesting that LexA binding reduces the affinity of TrpR for trpO by blocking the one-dimensional diffusion of TrpR.…”

“…Moreover, the effect also disappeared in vivo by blocking the diffusion by the introduction of a LexA site near trpO. 8 Our interpretation of the antenna effect of TrpR-trpO binding was challenged as a violation of the detailed balance of the rate equation. However, this criticism mistakenly supposes that detailed balance is a general truth, but this balance is actually an assumption required to describe a reaction with a single set of rate equations.…”

“…Moreover, the effect also disappeared in vivo by blocking the diffusion by the introduction of a LexA site near trpO . 8…”

“…9 If there is a degree(s) of freedom with a timescale close to or slower than that of the reaction, a conformational change, for example, detailed balance, no longer holds. [7][8][9][10][11] In this case, the reaction is described with two or more sets of rate equations switching alternatively for each reactant molecule according to the degree(s) of freedom. We named this mechanism the chemical ratchet because it generates a circulating stationary flow among the free components, nonspecific complex and TrpR-trpO specific complex in this case.…”

The Limitation of the Combination of Transition State Theory and Thermodynamics for the Reactions of Proteins and Nucleic Acids

“…The stationary state thus includes microscopic non-equilibrium as the switchings, and the absence of a microscopic equilibrium makes the rule of detailed balance indifferent. This is the case observed for the binding between E. coli TrpR and trpO , and the mechanism was named chemical ratchet [ 12 , 13 , 14 ].…”