Interconvertible Lac Repressor–DNA Loops Revealed by Single-Molecule Experiments

At many promoters, transcription is regulated by simultaneous binding of a protein to multiple sites on DNA, but the structures and dynamics of such transcription factor-mediated DNA loops are poorly understood. We directly examined in vitro loop formation mediated by Escherichia coli lactose repressor using single-molecule structural and kinetics methods. Small (∼150 bp) loops form quickly and stably, even with out-of-phase operator spacings. Unexpectedly, repeated spontaneous transitions between two distinct loop structures were observed in individual protein–DNA complexes. The results imply a dynamic equilibrium between a novel loop structure with the repressor in its crystallographic “V” conformation and a second structure with a more extended linear repressor conformation that substantially lessens the DNA bending strain. The ability to switch between different loop structures may help to explain how robust transcription regulation is maintained even though the mechanical work required to form a loop may change substantially with metabolic conditions. : Some proteins that regulate DNA transcription do so by binding simultaneously to two separated sites on the DNA molecule, forming a DNA loop. Although such loops are common, many of their features are poorly characterized. Of particular interest is the question of how some proteins accommodate the formation of loops of different sizes, particularly when the loops are small and thus require strong bending (and, in some cases, twisting) of the DNA to form. We observed the shape and behavior of individual DNA molecules bent into tight loops by Lac repressor, a transcription-regulating protein from the bacterium Escherichia coli. Loops were formed in DNA molecules with repressor-binding sites on opposite faces of the DNA double helix almost as readily as in those with sites on the same side, suggesting that the repressor is highly flexible. The DNA can switch back and forth between a tighter and a looser loop structure “on the fly” during the lifetime of a single loop, further evidence that Lac repressor is capable of adopting different shapes that may serve to minimize DNA bending or twisting in loops. The ability of the repressor to readily switch between different loop shapes may allow it to maintain effective control of transcription across situations in which the difficulty of bending or twisting DNA changes substantially. A large-scale conformational change in a transcription factor protein allows DNA loops to dynamically switch between alternative conformations that may contribute to robust transcription regulation.