DOWN-REGULATION OF ESCHERICHIA-COLI YFID EXPRESSION BY FNR OCCUPYING A SITE AT -93.5 INVOLVES THE AR1-CONTAINING FACE OF FNR

The promoter of the FNR-activated yfiD gene of Escherichia coli has an unusual architecture because it contains two FNR sites, an arrangemen t usually associated with FNR-mediated repression, Investigation of yf iD promoter derivatives with altered FNR sites revealed that occupatio n of the far upstream FNR site (FNR II) downregulated expression, desp ite the presence of a FNR dimer activating expression from the promote r proximal site (FNR I). Transcript mapping by primer extension, and m utagenesis of potential -10 elements, indicated that yfiD expression i s driven from a single FNR-dependent promoter with FNR sites at -40.5 (FNR I) and -93.5 (FNR II), However, yfiD mRNA is processed in station ary-phase cultures independently of me, rpoS, ihfA and fis to yield tr anscripts lacking 12 and 21 bases from their respective 5' ends, Singl e amino acid substitutions (G74-->C, F92-->S, A95-->P, R184-->P, P188- ->A or L193-->P) in the surface of FNR that contains activating region 1 (AR1 contacts the cn-subunit of RNA polymerase to promote transcrip tion activation) reduced the inhibitory effect of FNR at FNR II, indic ating that this region of the protein may have a role in repression as well as activation. The FNR variant F92-->S was notable because, alth ough it activated transcription of yfiD (two FNR sites), it was unable to activate transcription from model Class I and II promoters, which contain only a single FNR site.