The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding.

We have tagged the replication initiator protein of the plasmid R6K near the C-terminal end by fusion, in the correct reading frame, with the 89 amino acid long N-terminal alpha-donor polypeptide of beta-galactosidase of E. coli. This fusion was carried out with recombinant DNA methods. The protein chimera thus generated retained the activities of both initiation of DNA replication in vivo at the replication origin gamma of R6K and hydrolysis of beta-galactopyranoside when complemented in vivo with the alpha-acceptor polypeptide coded by the lac Z gene containing the M15 deletion. Using the simple and convenient assay for detecting beta-galactosidase, we have partially purified the tagged replication initiator, and have demonstrated that the protein binds to specific DNA sequences of the R6K chromosome. The protein bound to DNA sequences located at two places in the 5' untranslated leader region of the initiator protein cistron.