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Free Content Downregulation of Escherichia coli yfiD expression by FNR occupying a site at −93.5 involves the AR1-containing face of FNR

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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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Document Type: Original Article

Affiliations: Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.

Publication date: August 1, 1998

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