Autoregulation by cooperative binding of the PemI and PemK proteins to the promoter region of the pem operon.

The low copy number plasmid R100 carries the pem region, consisting of two genes, pemI and pemK, which are required for stable maintenance of the plasmid. Here, to understand the regulation of the expression of the pem region, we constructed plasmids carrying either the pemI or the pemK gene, whose initiation codons were fused in frame with the lacZ gene, and examined their expression by assaying beta-galactosidase (LacZ) activity. The synthesis of both PemI and PemK proteins was found to be repressed coordinately in the presence of a plasmid carrying the entire pem region. This indicates that pemK and pemI cistrons form an operon, and that the expression of the operon is negatively regulated by its own products. We then conducted a gel retardation assay in vitro and found that the two pem products, each of which was obtained as a tripartite protein (PemI-collagen-LacZ and PemK-collagen-LacZ), bound cooperatively to a specific fragment containing the proximal region of the pem operon. The binding region, determined by DNase I footprinting analysis, included the promoter for the pem operon. This indicates that both PemI and PemK proteins bind to the promoter region to autoregulate their synthesis.