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
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.