Single-Molecule In Vitro and Live-Cell Studies of Allostery through DNA

Abstract

Often in nature, a macromolecule undergoes conformational changes upon binding of a ligand in order to modify its affinity for another ligand at a distant binding site. This phenomenon, called “allostery”, is a fundamental mechanism for dynamic regulation of macromolecular properties. Although allostery is well-documented in proteins, it is less recognized for DNA-protein interactions, in which DNA has been often considered a mere template providing recognition sequences for proteins. Here we investigate the allosteric interactions through DNA both in single molecule experiments in vitro and in live cells. In the in vitro experiments, we demonstrate that when two proteins specifically bind to DNA within tens of base pairs, the binding affinity of one protein is altered by the other. We prove that this is not due to protein-protein interactions but to allostery through DNA. As the distance between the two proteins is varied, this allosteric coupling oscillates between positive and negative cooperativity with a periodicity of ∼10 base pairs, the helical pitch of the B-form DNA. The allostery through DNA is explained in terms of the free energy associated with the overall conformation of the ternary complex. We also demonstrate that such allostery affects gene expression in live E. coli cells, suggesting its physiological relevance.