Identification of a surface of FNR overlapping activating region 1 that is required for repression of gene expression

A library of Escherichia coli fnr mutants has been screened to identify FNR (regulator of fumarate and nitrate reduction) variants that are defective repressors, but competent activators. All but one of seventeen variants had substitutions close to or within the face of FNR that contains activating region 1 (AR1). Activating region 1 is known to contact the alpha subunit of RNA polymerase to facilitate transcription activation. It is now evident that this face also has a role in FNR-mediated repression. Single amino acid substitutions at Lys(54), Gly(74), Ala(95), Met(147), Leu(193), Arg(197), or Leu(239), and double substitutions at Ser(13) and Ser(145), Cys(16) and Ile(45), Tyr(69) and Ser(133), or Lys(164) and Phe(191), impaired FNR-mediated repression of ndh without greatly affecting activation from model Class I (FNR site at -71.5) and Class II (FNR site at -41.5) FNR-activated promoters. Although repression was impaired in a second group of FNR variants with substitutions at Leu(34), Arg(7) and Leul(193), Phe(92), or Ser(178), transcription activation from the simple FNR-dependent promoters was severely reduced. However, expression from pyfiD (FNR sites at -40.5 and -93.5) and a derivative lacking the site at -93.5, pyfiD-/+, remained relatively high indicating that this second group have a context-dependent activation defect as well as a repression defect. The prediction that the substitutions affecting repression were likely to be in solvent exposed regions of FNR was supported by analysis of peptides produced by partial proteolysis of FNR. Thus, FNR-mediated repression at promoters with multiple FNR sites requires regions of FNR that are different from, but overlap, AR1.