LacI-DNA-IPTG Loops: Equilibria among Conformations by Single-Molecule FRET

Abstract

The E. coli Lac repressor (LacI) tetramer binds simultaneously to a promoter-proximal DNA binding site (operator) and an auxiliary operator, resulting in a DNA loop, which increases repression efficiency. Induction of the lac operon by allolactose reduces the affinity of LacI for DNA, but induction does not completely prevent looping in vivo. Our previous work on the conformations of LacI loops used a hyperstable model DNA construct, 9C14, that contains a sequence directed bend flanked by operators. Single-molecule fluorescence resonance energy transfer (SM-FRET) on a dual fluorophore-labeled LacI-9C14 loop showed that it adopts a single, stable, high-FRET V-shaped LacI conformation. Ligand-induced changes in loop geometry can affect loop stability, and the current work assesses loop population distributions for LacI-9C14 complexes containing the synthetic inducer IPTG. SM-FRET confirms that the high-FRET LacI-9C14 loop is only partially destabilized by saturating IPTG. LacI titration experiments and FRET fluctuation analysis suggest that the addition of IPTG induces loop conformational dynamics and re-equilibration between loop population distributions that include a mixture of looped states that do not exhibit high-efficiency FRET. The results show that repression by looping even at saturating IPTG should be considered in models for regulation of the operon. We propose that persistent DNA loops near the operator function biologically to accelerate rerepression upon exhaustion of inducer.