N-TERMINAL TRUNCATED FORMS OF THE BIFUNCTIONAL PI-INITIATION PROTEIN EXPRESS NEGATIVE ACTIVITY ON PLASMID R6K REPLICATION

The replication initiation protein π 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 by direct repeats within the γ-origin region. By deleting C-terminal segments of the π coding region, we have found that the N-terminal polypeptides of π 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 π 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 γ-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 π 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 π protein. These findings demonstrate that the negative domain of π resides in the N-terminal segment of the protein. Furthermore, the data obtained suggest that inhibition of R6K replication by π does not require direct binding to DNA. © 1990 Springer-Verlag.