Replication from one of the three origins of the plasmid R6K requires coupled expression of two plasmid-encoded proteins.
The minimal beta-replicon of plasmid R6K contains an open reading frame for a 151-amino acid protein in addition to the seven 22-base pair direct nucleotide sequence repeats, the structural gene (pir) for the pi initiation protein, and the beta-origin sequence that have been shown to be required for replication activity. In this work, a site-specific mutation by linker insertion in this putative coding sequence, designated bis, resulted in a nonfunctional beta-replicon. The nonfunctional beta-replicon was complementable in trans and the protein coded by the bis sequence was detected in an immunoblot assay as a hybrid product from a bis-lac z fused gene. The bis gene is not required for a functional alpha or gamma origin replication origin of R6K. A site-specific mutation in the upstream pir gene was shown to lead to a loss of synthesis of the bis product and inactivation of the beta-replicon. Trans-complementation of this mutation for beta-replicon activity required the wild-type sequence of the pir gene joined to the intact bis sequence. These results indicate that the bis product is required for activity specifically of the beta-origin, and its synthesis is coupled in cis to the expression of pi protein from an unaltered pir gene.
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004261:DNA Replication; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D011815:R Factors; D012093:Replicon; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D001616:beta-Galactosidase