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Conjugative DNA transfer is a highly conserved process for the direct transfer of DNA from a donor to a recipient. The conjugative initiator proteins are key players in the DNA processing reactions that initiate DNA transfer – they introduce a site- and strand-specific break in the DNA backbone via a transesterification that leaves the initiator protein covalently bound on the 5′-end of the cleaved DNA strand. The action of the initiator protein at the origin of transfer (oriT) is governed by auxiliary proteins that alter the architecture of the DNA molecule, allowing binding of the initiator protein. In the F plasmid system, two auxiliary proteins have roles in establishing the relaxosome: the host-encoded IHF and the plasmid-encoded TraY. Together, these proteins direct the loading of TraI which contains the catalytic centre for the transesterification. The F-oriT sequence includes a binding site for another plasmid-encoded protein, TraM, which is required for DNA transfer. Here the impact of TraM protein on the formation and activity of the F plasmid relaxosome has been examined. Purified TraM stimulates the formation of relaxed DNA in a reaction that requires the minimal components of the relaxosome, TraI, TraY and IHF. Unlike TraY and IHF, TraM is not essential for the formation of the relaxosome in vitro and TraM cannot substitute for either TraY or IHF in this process. The TraM binding site sbmC, along with both IHF binding sites, is essential for stimulation of the relaxase reaction. In addition, stimulation of transesterification appears to require the C-terminal domain of TraI suggesting that TraM and TraI may interact through this domain on TraI. Taken together, these results provide additional evidence of a role for TraM as a component of the relaxosome, suggest a previously unknown interaction between TraI and TraM, and allow us to propose a molecular role for the C-terminal domain of TraI.