title: FNR-dependent repression of ndh gene expression requires two upstream FNR-binding sites.
abstract: The ndh gene of Escherichia coli encodes a non-proton-translocating NADH dehydrogenase (NdhII) that is anaerobically repressed by the global transcription regulator, FNR. FNR binds at two sites (centred at -50.5 and -94.5) in the ndh promoter but the mechanism of FNR-mediated repression appears not to be due to promoter occlusion. This mechanism has been investigated using an aerobically active derivative of FNR, FNR* (FNR-D154A), with ndh promoters containing altered FNR-binding sites. FNR* repressed ndh gene expression both aerobically and anaerobically in vivo. Gel retardation analysis and DNase I footprinting with purified FNR* protein confirmed that FNR interacts at two sites in the ndh promoter, and that FNR and RNA polymerase (RNAP) can bind simultaneously. Studies with three altered ndh promoters, each containing an impaired or improved FNR-site, indicated that both FNR-sites are needed for efficient repression in vivo. The alpha-subunit of RNAP interacted with two regions (centred at -105 and -46), each overlapping one of the FNR-sites in the ndh promoter. Footprints of the FNR*-RNAP-ndh ternary complex indicated that FNR*-binding at -50.5 prevents the alpha-subunit of RNAP from docking with the DNA just upstream of the -35 element. Binding of a second FNR* molecule at the -105 site likewise prevents binding of the alpha-subunit at its alternative site, thus providing a plausible mechanism for FNR-mediated repression based on displacement of the alpha-subunit of RNAP.
authors.affiliation: The Koreas Institute for Biomolecular Research, Department of Molecular Biology & Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Wenmao
authors.initials: W
authors.lastname: Meng
authors.affiliation: The Koreas Institute for Biomolecular Research, Department of Molecular Biology & Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Green
authors.affiliation: The Koreas Institute for Biomolecular Research, Department of Molecular Biology & Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
authors.forename: John R
authors.initials: JR
authors.lastname: Guest
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D007506:Iron-Sulfur Proteins / Q000502:physiology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009245:NADH Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; C088908:NADH dehydrogenase II; D009245:NADH Dehydrogenase; D012321:DNA-Directed RNA Polymerases