Plasmid pLS1-encoded RepA protein regulates transcription from repAB promoter by binding to a DNA sequence containing a 13-base pair symmetric element

Abstract

The repA gene product of the promiscuous plasmid pLS1 is a 45-amino acid repressor protein. The plasmid initiator of replication protein, RepB, is encoded by the repB gene which is situated downstream of repA. The results presented here demonstrate that both genes constitute a transcriptional unit. We show that the repA gene product inhibits transcription from the repAB promoter both in vitro and in vivo. By hydroxyl radical footprinting on both DNA strands, we show that RepA binds specifically to a plasmid region in which a 13-base pair element, showing a 2-fold rotational symmetry, is located. Within this symmetric element lies the -35 region of the repAB promoter. RepA binds into successive major grooves along one face of the DNA helix. The general architecture of RepA and of its interactions with DNA resembles that of the Cro repressor proteins of bacteriophages lambda and 434. We propose that RepA regulates the plasmid copy number by binding to its own promoter, thus controlling the synthesis of the plasmid initiator of replication protein.