GapR is a nucleoid-associated protein that stiffens and overtwists DNA to promote transcription and replication
GapR is an -proteobacterial nucleoid-associated protein 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, while promoting duplex DNA melting at higher 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 differentiate between AT-rich and overtwisted DNA binding, revealing that overtwisted DNA primarily determines GapR localization. Lastly, we show that these two binding modes exert opposite effects on transcription. AT-rich binding stimulates initiation, likely by promoting RNA polymerase progression, while overtwisted DNA binding represses transcription, possibly by stabilizing local overtwist at promoters. We propose that GapR binding promotes a conformation of DNA ideal for efficient replication and transcription progression. 3 Key pointsO_LIGapR is a DNA structuring protein that binds AT-rich and overtwisted, positively supercoiled DNA C_LIO_LIGapR binding stiffens DNA, limits plectoneme formation, and melts undertwisted DNA at higher force C_LIO_LIGapR binding modes exert opposing effects on transcription initiation while promoting elongation C_LI