Data for Li et al FEBS Letters 2024

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
(TraICTD) wraps around TraK’s C-proximal tetramerization domain. Upon
relaxosome assembly, conformational changes disrupt this contact, and
TraICTD 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.