Characterization of OmpR binding sequences in the upstream region of the ompF promoter essential for transcriptional activation

Abstract

In Escherichia coli the expression of the outer membrane porin gene ompF requires the transcriptional activator protein OmpR. Previous DNase I footprinting experiments with purified OmpR localized the OmpR binding site from positions -105 to -60 (relative to the transcriptional start site) in the ompF promoter, and three tandem 10-base pair sequences elements (Fa, Fb, and Fc) within this region were proposed to be important for OmpR recognition. In order to elucidate the roles of the F boxes for transcriptional activation of ompF, various F box deletions and point mutations were constructed and analyzed for their effects on ompF-lacZ expression and OmpR binding. Removal of 102 nucleotides, which included a portion of the OmpR binding region (the Fa box), evidenced the largest decrease in transcriptional activation and significantly reduced OmpR binding. Additional deletion of four more base pairs in this target site (representing half of the Fb box) further reduced ompF expression. OmpR interactions with DNA sequences representing the OmpR binding region were analyzed by DNA mobility shift experiments. A 43-base pair ompF oligonucleotide containing the Fa, Fb, and Fc regions was sufficient for OmpR-dependent DNA binding using either purified OmpR or cell supernatants. The central C residue in each F box was changed to a T and unique patterns of protein-DNA complexes were observed that were different from that of the wild type binding site. The most dramatic effect on OmpR binding was observed when the C to T transversion occurred in the Fb box, and this mutation also reduced the level of ompF-lacZ expression. Our results indicate that the F boxes play important roles in the activation of ompF expression, and we suggest that OmpR may interact cooperatively with these boxes.