FNR-dependent activation of the class II dmsA and narG promoters of Escherichia coli requires FNR-activating regions 1 and 3

In Escherichia coil, the anaerobic expression of genes encoding the nitrate (narGHJI) and dimethyl sulphoxide (dmsABC) terminal reductases is stimulat ed by the global anaerobic regulator FNR. The ability of FNR to activate tr anscription initiation has been proposed to be dependent on protein-protein interactions between RNA polymerase and two activating regions (AR) of FNR , FNR-AR1 and FNR-AR3, To further our understanding of the role of FNR-AR1 and FNR-AR3 in transcription activation, we measured the effects of FNR-AR mutants on expression of the narG and dmsA promoters, P-narG and P-dmsA All the FNR-AR1 (FNR-S73F, FNR-T118A, FNR-S187P), FNR-AR3 (FNR-G85A) and FNR-A R1-AR3 (FNR-G85A-S187P) mutants that were tested decreased expression from P-narG and P-dmsA in vivo. Transcription assays of P-dmsA also showed that the FNR-AR mutant proteins impaired transcription activation in vitro, Furt hermore, DNase I footprinting analysis confirmed that this transcription de fect was not a result of altered DNA-binding properties. The function of FN R-S187P and FNR-G85A was also measured in strains containing sigma (70) mut ants (sigma (70)-K593A, sigma (70)-R596A and sigma (70)-K597A) known to be impaired in FNR-dependent transcription activation. Of all of the combinati ons analysed, only FNR-G85 and sigma (70)-K597 showed a genetic interaction , supporting the notion that FNR-AR3 and sigma (70) interact functionally i n the process of transcription activation, Lastly, the transcription activa tion defect of the FNR-AR1 and FNR-AR3 mutants was greatly reduced when exp ression of P-narG was assayed in the presence of nitrate, As these growth c onditions promote maximal activity of P-narG as a result of the combined fu nction of NarL, IHF and FNR, these results suggest that the requirements fo r FNR-AR1 and FNR-AR3 are altered in the presence of additional activators.