Negative control of plasmid DNA replication by iterons. Correlation with initiator binding affinity.

Initiation of DNA replication and negative control of initiation frequency in many bacterial plasmids are mediated by multiple binding sites (iterons) for the replicon-encoded initiator protein. Here we show that a single iteron of plasmid P1, when cloned in a multicopy vector, pUC19, can control replication of a miniP1 plasmid effectively. Using this assay system, several iterons with single point mutations were analyzed. The degree of control was directly correlated with the binding affinity of the iterons for the initiator protein in vitro. The control was also unaffected whether or not a wild type iteron was flanked with strong transcription terminators. We conclude that initiator binding is the only activity of P1 iteron sequences relevant to replication control.