TheEsherichia colitranscription factor FNR regulates expression of genes required for the metabolic switch between aerobic and anaerobic respiration. In order to investigate how FNR controls transcription of its target operons, DNA binding was examined for both the wild-type (WT) FNR protein and an altered function FNR* protein (DA154) that exhibits enhanced activity in the presence of oxygen bothin vivoandin vitro, apparently due to the fact that DA154 is able to dimerize to a greater extent than WT FNR. Electrophoretic mobility shift assays, using a consensus symmetrical FNR target site, revealed that both DA154 and WT FNR gave rise to protein-DNA complexes of indistinguishable electrophoretic mobilities. In addition, an estimate of the molecular weight from the mobility of the DA154-DNA complex indicated that both mutant and WT FNR were dimeric when bound to DNA. Under the same binding conditions, DA154 showed an observed constant of ~3x10 8 M - 1 for the consensus symmetrical target site. In addition, the results of DNA binding competition assays provided evidence that DA154 was a site-specific DNA binding protein, since this mutant protein bound to the consensus symmetrical target site with approximately 40-fold and 250-fold higher affinity than a natural target site from thenarpromoter or a non-specific DNA target, respectively. Electrophoretic mobility shift DNA bending assays demonstrated protein-induced DNA bending by both DA154 and WT FNR. In addition,in vitrotranscription assays using an FNR-depdendent variant of thelacP1 promoter demonstrated levels of transcription activation by DA154 comparable to those observedin vivo. These results provide several new insights into how FNR functions to activate transcription of target genes.