In preparation for transfer conjugative type IV secretion systems (T4SS) produce a nucleoprotein adduct containing a relaxase enzyme covalently linked to the 5' end of single-stranded plasmid DNA. The bound relaxase is expected to present features necessary for selective recognition by the type IV coupling protein (T4CP), which controls substrate entry to the envelope spanning secretion machinery. We prove that the IncF plasmid R1 relaxase TraI is translocated to the recipient cells. Using a Cre recombinase assay (CRAfT) we mapped two internally positioned translocation signals (TS) on F-like TraI proteins that independently mediate efficient recognition and secretion. Tertiary structure predictions for the TS matched best helicase RecD2 from Deinococcus radiodurans. The TS is widely conserved in MOB(F) and MOB(Q) families of relaxases. Structure/function relationships within the TS were identified by mutation. A key residue in specific recognition by T4CP TraD was revealed by a fidelity switch phenotype for an F to plasmid R1 exchange L626H mutation. Finally, we show that physical linkage of the relaxase catalytic domain to a TraI TS is necessary for efficient conjugative transfer.