Data for Li et al FEBS Letters 2024

Conjugative dissemination of mobile genetic elements (MGEs) among bacteriais initiated by assembly of the relaxosome at the MGE’s origin-of-transfer(oriT) sequence. A critical but poorly defined step of relaxosome assemblyinvolves recruitment of the catalytic relaxase to its DNA strand-specific nickingsite within oriT. Here, we present evidence by AlphaFold modeling, affinitypulldowns, and in vivo site-directed photocrosslinking that the TraKRibbon–Helix–Helix DNA-binding protein recruits TraI to oriT through adynamic interaction in which TraI’s C-terminal unstructured domain(TraICTD) wraps around TraK’s C-proximal tetramerization domain. Uponrelaxosome assembly, conformational changes disrupt this contact, andTraICTD instead self-associates as a prerequisite for relaxase catalytic functionsor substrate engagement with the transfer channel. These findings delineatekey early-stage processing reactions required for conjugativedissemination of a model MGE.