Downregulation ofEscherichia coli yfiDexpression by FNR occupying a site at −93.5 involves the AR1-containing face of FNR

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Abstract
The promoter of the FNR‐activated yfiD gene of Escherichia coli has an unusual architecture because it contains two FNR sites, an arrangement usually associated with FNR‐mediated repression. Investigation of yfiD promoter derivatives with altered FNR sites revealed that occupation of the far upstream FNR site (FNR II) downregulated expression, despite the presence of a FNR dimer activating expression from the promoter proximal site (FNR I). Transcript mapping by primer extension, and mutagenesis of potential −10 elements, indicated that yfiD expression is 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 stationary‐phase cultures independently of rne, rpoS, ihfA and fis to yield transcripts lacking 12 and 21 bases from their respective 5′ ends. Single 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 α‐subunit of RNA polymerase to promote transcription activation) reduced the inhibitory effect of FNR at FNR II, indicating that this region of the protein may have a role in repression as well as activation. The FNR variant F92→S was notable because, although 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.

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