Characterization by deletion mutagenesis in vitro of the promoter region of ompF, a positively regulated gene of Escherichia coli

Abstract

The ompF gene codes for a major outer membrane protein whose expression is positively regulated by the ompR and envZ genes. Two sets of promoter deletions, upstream deletions and downstream deletions, were generated in vitro, and the promoter function was studied by connecting them with the tet genes. One of the hybrid genes thus constructed had a functioning ompF-tet hybrid promoter. The 107 base-pair fragment was found to be functioning as the ompF promoter, 90 nucleotides upstream and 17 nucleotides downstream of the mRNA start site that was also determined in this study. The start site was preceded by a convenient Pribnow box. Although the sequence at the −35 region had a low degree of homology to the consensus sequence, analyses of the hybrid promoter suggested that this region is involved in the promoter function in relation to the Pribnow box. They also indicated that the domain responsible for regulation by the ompR gene is located within the −35 region and its upstream region.