Miscoordination of the iron-sulfur clusters of the anaerobic transcription factor, FNR, allows simple repression but not activation.
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.
Scott
Colin
C
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom.
Green
Jeffrey
J
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*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D014157:Transcription Factors