title: Miscoordination of the iron-sulfur clusters of the anaerobic transcription factor, FNR, allows simple repression but not activation.
abstract: The FNR protein of Escherichia coli regulates target genes in response to anaerobiosis. Environmental oxygen is sensed by the acquisition of oxygen-labile [4Fe-4S] clusters that promote dimerization, DNA binding, and productive interactions with RNA polymerase. Three N-terminal cysteine residues (Cys(20), Cys(23), and Cys(29)) and Cys(122) act as ligands for the FNR iron sulfur clusters. An FNR variant, FNR-C20S, that retains only trace activity in vivo can acquire [4Fe-4S] clusters in vitro that enhance site-specific DNA binding. Second site substitutions in activating regions AR1, AR2, and AR3 restore in vivo activity to FNR-C20S, suggesting that the impairment in FNR-C20S activity is due to a failure to communicate with RNA polymerase effectively. Here we show that FNR-C20S can repress a simple FNR-regulated promoter in vivo and that it can form productive heterodimers with an FNR variant with altered DNA binding specificity, FNR-E209V. Transcription studies with FNR-E209V.FNR-C20S heterodimers indicate that the presence of a miscoordinated iron-sulfur cluster (FNR-C20S) in the downstream (but not the upstream) subunit of the FNR dimer impairs activation from a class II promoter and that this impairment can be overcome by amino acid substitutions known to unmask AR2 or improve AR3 in the affected subunit.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom.
authors.forename: Colin
authors.initials: C
authors.lastname: Scott
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Green
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000502:physiology*; D009154:Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D014157:Transcription Factors