RepA protein- and oriR-dependent initiation of R1 plasmid replication: identification of a rho-dependent transcription terminator required for cis-action of repA protein.

Initiation of R1 plasmid replication is dependent on cis-acting repA protein and the 188 base-pair (bp) sequence, oriR. RepA protein synthesized in vitro preferentially activates oriR in cis, regardless of the orientation and location of oriR on the template DNA. RepA protein is not reusable after it activates oriR in the cis-position. Cis-action of repA protein is also dependent on the presence of CIS, a 170 bp sequence, between repA and oriR. CIS contains a rho dependent transcription terminator of the repA transcript, deletion of which results in decrease in transformation efficiency and rapid loss of plasmid in the absence of selection. The significance of transcription termination events in replication was indicated by decreased replication activity in vivo caused by premature termination of the repA transcript between repA and CIS. A model which may account for the role of CIS in mediating the cis-action of the repA protein is presented.