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

Takashi Kinebuchi,Nobuo Shimamoto

doi:10.1038/s41598-021-83156-6

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.

Highlights

The antenna effect for a protein with single binding site, E. coli EcoRI methyltransferase, was found by Surby and Reich[10] as the first systematic study of the effect
Since the affinity is determined at equilibrium, the rule seemingly denies the antenna effect caused by onedimensional diffusion
We found that the “equilibrium” required for detailed balance is different than the one required for determining an affinity

Summary

Introduction

The antenna effect for a protein with single binding site, E. coli EcoRI methyltransferase, was found by Surby and Reich[10] as the first systematic study of the effect. By using gel-shift assay, they showed that the affinity for whole DNA fragments increased 20-fold as the length increased from 14 to 775 bp, while the observed dissociation rate constant was independent of the length From the results, they proposed a mechanism based on the accelerated association by one-dimensional diffusion along DNA with a declared reservation of the “violation of the thermodynamic rule”, without further comment. They proposed a mechanism based on the accelerated association by one-dimensional diffusion along DNA with a declared reservation of the “violation of the thermodynamic rule”, without further comment This rule is usually called detailed balance and prohibits the existence of net circulation flow among reaction components at equilibrium, and claims that acceleration of a rate must be accompanied by the acceleration of its reverse rate of every step in the equilibrated reaction, maintaining the binding affinity for the specific site unchanged. The measured expression of lacZ showed a good correlation to the LexA affinity for the sites, providing evidence for the existence of one-dimensional diffusion as well as antenna effect due to the diffusion in vivo, suggesting the cross-talk between two proteins at a distance on DNA

Methods

Results

Conclusion