Reconstitution of the [4Fe-4S] cluster in FNR and demonstration of the aerobic-anaerobic transcription switch in vitro.
The FNR protein of Escherichia coli is a redox-responsive transcription regulator that activates and represses a family of genes required for anaerobic and aerobic metabolism. Reconstitution of wild-type FNR by anaerobic treatment with ferrous ions, cysteine and the NifS protein of Azotobacter vinelandii leads to the incorporation of two [4Fe-4S]2+ clusters per FNR dimer. The UV-visible spectrum of reconstituted FNR has a broad absorbance at 420 nm. The clusters are EPR silent under anaerobic conditions but are degraded to [3Fe-4S]+ by limited oxidation with air, and completely lost on prolonged air exposure. The association of FNR with the iron-sulphur clusters is confirmed by CD spectroscopy. Incorporation of the [4Fe-4S]2+ clusters increases site-specific DNA binding about 7-fold compared with apo-FNR. Anaerobic transcription activation and repression in vitro likewise depends on the presence of the iron-sulphur cluster, and its inactivation under aerobic conditions provides a demonstration in vitro of the FNR-mediated aerobic-anaerobic transcriptional switch.
Green
J
J
The Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, U.K.
Bennett
B
B
Jordan
P
P
Ralph
E T
ET
Thomson
A J
AJ
Guest
J R
JR
D000332:Aerobiosis; D000693:Anaerobiosis; D016948:Azotobacter vinelandii / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000494:pharmacology; D004269:DNA, Bacterial / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D013053:Spectrophotometry; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D014157:Transcription Factors; C080001:nifS protein, Bacteria; D003545:Cysteine