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. 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. SM-FRET confirms that the high-FRET LacI-9C14 loop is only partially destabilized by saturating IPTG. FRET histograms suggest that the remaining population is a mixture of lower-FRET states ascribed to specific nonspecific or extended LacI loops, not free DNA.