ISOLATION OF AN OXYGEN-SENSITIVE FNR PROTEIN OF ESCHERICHIA-COLI - INTERACTION AT ACTIVATOR AND REPRESSOR SITES OF FNR-CONTROLLED GENES

Citation
Sb. Melville et Rp. Gunsalus, ISOLATION OF AN OXYGEN-SENSITIVE FNR PROTEIN OF ESCHERICHIA-COLI - INTERACTION AT ACTIVATOR AND REPRESSOR SITES OF FNR-CONTROLLED GENES, Proceedings of the National Academy of Sciences of the United Statesof America, 93(3), 1996, pp. 1226-1231
Citations number
27
Categorie Soggetti
Multidisciplinary Sciences
ISSN journal
00278424
Volume
93
Issue
3
Year of publication
1996
Pages
1226 - 1231
Database
ISI
SICI code
0027-8424(1996)93:3<1226:IOAOFP>2.0.ZU;2-Z
Abstract
The Escherichia coli fnr gene product, FNR, is a DNA binding protein t hat regulates a large family of genes involved in cellular respiration and carbon metabolism during conditions of anaerobic cell growth, FNR is believed to contain a redox/O-2-sensitive element for detecting th e anaerobic state. To investigate this process, a fnr mutant that enco des an altered FNR protein with three amino acid substitutions in the N-terminal domain was constructed by site-directed mutagenesis, In viv o, the mutant behaved like a wild-type strain under anaerobic conditio ns but had a 14-fold elevated level of transcriptional activation of a reporter gene during aerobic cell growth, The altered fnr gene was ov erexpressed in E, coil and the resultant FNR protein was purified to n ear homogeneity by using anaerobic chromatography procedures, An in vi tro Rsa I restriction site protection assay was developed that allowed for the assessment of oxygen-dependent DNA binding of the mutant FNR protein, The FNR protein was purified as a monomer of M(r) 28,000 that contained nonheme iron at 2.05 +/- 0.34 mot of Fe per FNR monomer, In vitro DNase I protection studies were performed to establish the loca tions of the FNR-binding sites at the narG, narK, dmsA, and hemA promo ters that are regulated by either activation or repression of their tr anscription, The sizes of the DNA footprints are consistent with the b inding of two monomers of FNR that protect the symmetrical FNR-recogni tion sequence TTGAT-nnnnATCAA. Exposure of the FNR protein or protein- DNA complex to air for even short periods of time (approximate to 5 mi n) led to the complete loss of DNA protection at a consensus FNR recog nition site, A model whereby the FNR protein exists in the cell as a m onomer that assembles on the DNA under anaerobic conditions to form a dimer is discussed.