Conjugative transfer of the <scp>I</scp> nc <scp>N</scp> plasmid p <scp>KM</scp> 101 is mediated by dynamic interactions between the <scp>T</scp> ra <scp>K</scp> accessory factor and <scp>T</scp> ra <scp>I</scp> relaxase

Conjugative dissemination of mobile genetic elements (MGEs) among bacteria is initiated by assembly of the relaxosome at the MGE's origin-of-transfer (oriT) sequence. A critical but poorly defined step of relaxosome assembly involves recruitment of the catalytic relaxase to its DNA strand-specific nicking site within oriT. Here, we present evidence by AlphaFold modeling, affinity pulldowns, and in vivo site-directed photocrosslinking that the TraK Ribbon-Helix-Helix DNA-binding protein recruits TraI to oriT through a dynamic interaction in which TraI's C-terminal unstructured domain (TraI_CTD) wraps around TraK's C-proximal tetramerization domain. Upon relaxosome assembly, conformational changes disrupt this contact, and TraI_CTD instead self-associates as a prerequisite for relaxase catalytic functions or substrate engagement with the transfer channel. These findings delineate key early-stage processing reactions required for conjugative dissemination of a model MGE.