Relaxase asymmetry drives initiation of bacterial conjugation

Abstract Bacterial conjugation drives horizontal gene transfer and accelerates the spread of antibiotic resistance. In F-plasmid systems, the bifunctional relaxase-helicase TraI initiates transfer by nicking plasmid DNA through its trans-esterase activity and subsequently unwinding the plasmid via its helicase domains. Previous evidence suggested that two TraI molecules act cooperatively, yet how their activities are spatially and temporally coordinated has remained unclear. Here, we report the cryo-EM structure of a functionally asymmetric TraI homodimer and show that dimerisation is mediated predominantly by single-stranded DNA. Trans-esterasedriven duplex melting at the origin of transfer by one TraI molecule enables helicase loading by a second TraI molecule onto the transfer strand of the intact plasmid. Although the trans-esterase domain is intrinsically active, the host factor IHF inhibits nicking until helicase loading occurs. These findings uncover a regulatory loop coupling strand nicking with helicase engagement, defining a unified mechanism for initiating conjugative DNA processing.