Coordinated repression in vitro of the divergent fepA-fes promoters of Escherichia coli by the iron uptake regulation (Fur) protein

Abstract

The mechanism involved in transcriptional repression of the fepA-fes divergent promoters of Escherichia coli by the Fur (ferric uptake regulation) protein has been examined in vitro. This DNA region includes a suboptimal and single Fur-binding site with two divergent and overlapped -35/-10 hexamers. Comparison of transcription patterns generated with runoff experiments in either the presence or the absence of heparin showed that access of the RNA polymerase to the principal -35/-10 hexamers was fully prevented by Fur- Mn2+ bound to its target site within the divergent promoter region. Neither RNA polymerase bound to the fes and fepA promoters could be displaced by Fur- Mn2+, nor could the bound repressor be outcompeted by an excess of the enzyme. However, the repressor blocked reinitiation as soon as the polymerase moved away from the fes promoter during transcription. The spatial distribution of regulatory elements within the DNA region allowed the simultaneous binding of the RNA polymerase to the fes and fepA promoters and their coordinate regulation regardless of their different transcriptional activities. Comparison with other iron-regulated systems support a general mechanism for Fur-controlled promoters that implies a direct competition between the polymerase and the regulator for overlapping target sites in the DNA.