GapR stiffens AT-rich and overtwisted DNA leading to differential effects on transcription based on gene context.

GapR is a pleiotropic α-proteobacterial nucleoid-associated protein (NAP) reported to either directly regulate transcription of AT-rich DNA or to regulate transcription indirectly through sensing DNA topology and modulating topoisomerase activity. We use single-DNA micromechanics, biolayer interferometry (BLI), and computational analysis to study GapR transcriptional regulation. Micromechanics experiments show that GapR overtwists DNA and shortens its contour length. GapR binding also substantially increases DNA bending persistence length and twist stiffness. For DNA tension ∼0.5 pN, as occurs in supercoiled domains, GapR also promotes DNA strand separation, a transition not observed for lower forces. Nonequilibrium binding experiments show GapR-DNA complexes to be extremely stable, with essentially no dissociation on hour-long time scales. Strikingly, our BLI experiments show that GapR has high affinity for AT-rich DNA, while our micromechanics show that GapR binding is enhanced by pre-twisting of DNA, validating GapR affinity for both forms. By analyzing published GapR binding data, we reveal that overtwisted DNA primarily determines GapR localization. We demonstrate that these two binding modes have opposing impacts on gene expression, with AT-rich binding activating and overtwisted DNA binding repressing transcription. Together, our findings demonstrate how the biophysical activities and topological sensitivity of a single NAP generate context-specific behavior.