Promoter sequence requirements for Fnr-dependent activation of transcription of the narGHJI operon

The sequence requirements for Fnr-dependent transcription of the narGHJI operon of Escherichia coli were studied in plasmids carrying a narG::lacZ protein fusion with the 5' end of the promoter deleted so that expression was controlled exclusively by Fnr. These plasmids were subjected to in vitro mutagenesis, and beta-galactosidase activities were determined in transformed strains after aerobic and anaerobic growth. A single base-pair change in the Fnr box, a sequence which is highly conserved in all Fnr-dependent promoters, essentially abolished anaerobic induction of expression. Primer extension analysis located the transcription start site 57 bp upstream from the narG translation start site and placed the Fnr box at a position centred between -41 and -42 bases from the transcription start site. The position of the Fnr box relative to the transcription start site was critical for anaerobic induction of expression. The deletion of 2 bp or addition of 4, 6, 10, 14, 20, 22, 28, 30, and 40 bp immediately downstream from the Fnr box abolished anaerobic induction. Sequence changes between the Fnr box and the transcription start site had different effects, depending upon the region mutagenized. Base changes immediately downstream from the Fnr box, including bases -20 to -29, did not lead to any decrease in anaerobic induction of expression, but in most instances resulted in increased expression. Base changes further downstream prevented anaerobic induction of expression and suggested the requirement for a -10 hexamer which is partially homologous to the -10 consensus sequence for sigma 70-specific promoters of E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)