Abstract
The replication control system of plasmid R6-5 has been investigated by characterization of high-copy-number mutant miniplasmids, development of an in vivo assay for the site of action or "target" of the replication control elements, and sequence analysis of the replication control regions of the wild-type plasmid and two copy-number mutant derivatives. These and other experiments have shown that three plasmid determinants--copA/incA, copB, and copT--are involved in DNA replication control. The products of the copB and copA/incA genes, a 9500-dalton basic polypeptide and either a 7200-dalton basic polypeptide or a short untranslated RNA molecule, respectively, are negative-acting elements that interact with the third element, their target, the copT DNA sequence, or its product to regulate the frequency of initiation of plasmid replication. The location of copT within the copA/incA gene and 1600 base pairs upstream from the origin of replication indicates that regulation is effected at a preinitiation stage of replication, such as the production of a primer or other initiation factor.