THE PLASMID RK2 INITIATION PROTEIN BINDS TO THE ORIGIN OF REPLICATIONAS A MONOMER

The TrfA protein encoded by the broad host range bacterial plasmid RK2 specifically binds to eight direct repeats (iterons) present at the p lasmid replication origin to initiate DNA replication. purified TrfA p rotein is largely in the form of a dimer, and using a dimerization tes t system that involves the fusion of the amino-terminal domain of the lambda cI repressor protein to TrfA, we show that the TrfA protein for ms dimers in vivo., Because of the high stability of the dimer form of TrfA, the formation of heterodimers between the wild-type and differe nt sized TrfA proteins requires in vivo de novo folding of the primary protein sequence or in vitro denaturation and renaturation. The resul ts of gel mobility shift assays using in vitro or in vivo formed heter odimers indicated that the TrfA protein binds to the iteron DNA as a m onomer. Furthermore, when the monomeric and dimeric forms of TrfA are separated by gel filtration chromatography, only the protein in the ch romatographic position of the monomeric form demonstrated significant DNA binding activity. These results indicate that only the monomer for m of the TrfA protein is active for binding to the iterons at the RK2 replication origin.