N-terminal truncated forms of the bifunctional pi initiation protein express negative activity on plasmid R6K...

The replication initiation protein pi of the Escherichia coli plasmid R6K is a dual regulator in the control of plasmid copy number, functioning both as a specific initiator and inhibitor of replication. While the biochemical basis of these activities is not known, initiator activity requires binding of the protein to the seven 22 bp direct repeats within the gamma-origin region. By deleting C-terminal segments of the pi coding region, we have found that the N-terminal polypeptides of pi that are produced, corresponding to the first 117 and 164 amino acids, respectively, retain the negative activity of the bifunctional protein, i.e. these truncated pi proteins specifically inhibit R6K replication in vivo. These negatively acting polypeptides, however, are incapable of initiating replication in vivo and fail to bind to the gamma-origin of the R6K DNA in vitro. A correspondence between the observed negative activity of the N-terminal peptide and the negative regulatory activity of the intact pi protein is supported by the finding that point mutations introduced into the 164 amino acid N-terminal peptide that result in a decrease in its inhibitory activity also produce a plasmid high-copy phenotype when these mutations are incorporated into the full-length pi protein. These findings demonstrate that the negative domain of pi resides in the N-terminal segment of the protein. Furthermore, the data obtained suggest that inhibition of R6K replication by pi does not require direct binding to DNA.