Allostery occurs when the binding of a ligand at one site of a protein causes conformational changes at a distant site. Such effects have been extensively studied for enzymes and other proteins, but there were also indications that allostery could occur in DNA. Now Xie, Sun, Su and colleagues have directly observed and characterized allosteric effects through DNA, using a single-molecule fluorescence microscopy approach (Science 339, 816–819, 2013).

The authors immobilized a DNA fragment containing binding sites for two different proteins, separated by a linker of variable length. The first protein was fluorescently labeled, so that its association with DNA could be visualized; then the second protein was added, and the dissociation of the first one was monitored.

The data revealed that the dissociation rate (koff) of the first protein was increased or decreased by the presence of the second protein (see illustration), in an oscillating manner, as a function of linker length and with a periodicity of 10 bp, which is the helical pitch of B-form DNA. The effect disappeared when the linker was longer than 25 bp, yielding a decay length of ∼15 bp. Similar results were obtained for DNA-binding proteins, with different sizes, shapes, surface electrostatic features or affinities for their DNA targets.

The oscillating effect was not sensitive to ionic strength conditions, ruling out contributions from potential protein-protein electrostatic interactions. On the other hand, distorting the structure of the linker region—with a nick or mismatch, or using GC-rich sequences—dampened the oscillation. Moreover, replacing the first protein-binding site with a DNA hairpin similarly affected the koff of a bound protein nearby. Notably, the amplitude of the oscillation was lower with a long-loop hairpin, which should cause a smaller DNA distortion, than with a short-loop hairpin. These observations suggest that DNA mechanical properties may produce the allosteric effect.

The authors then used molecular dynamic simulations to examine the structure of DNA, either free in solution or with its central base pair pulled apart to mimic the effect of a bound protein. In the latter condition, the major groove width was altered, compared to that of free DNA, in an oscillating manner and with a periodicity of ∼10 bp. Such deviations in major groove width would affect the stability of a DNA-bound protein nearby, accounting for the allosteric coupling between the two sites.

Finally, the DNA allosteric effect was seen in two biologically relevant systems, in both cases with the characteristic oscillatory pattern described above. The presence of a nearby nucleosome affected the binding of a glucocorticoid receptor DNA-binding domain to its target in vitro. The activity of T7 RNA polymerase on a promoter was affected by LacR bound nearby: detailed binding kinetics data were obtained in vitro and, importantly, similar effects were seen on transcriptional activity in bacterial cells.