Downregulation 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 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 alpha-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.
Green
J
J
Department of Molecular Biology and Biotechnology, University of Sheffield, UK. jeff.green@sheffield.ac.uk
Baldwin
M L
ML
Richardson
J
J
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D014157:Transcription Factors