FNR is a transcriptional regulator that controls gene expression in response to oxygen limitation in Escherichia coli. The NADH dehydrogenase II gene (ndh) is repressed by FNR under anaerobic conditions. Repression is not simply due to occlusion of the promoter (-35 and -10) region by FNR because adjacent pairs of FNR monomers were found to bind at two sites centred at -50.5 and -94.5 in the ndh promoter region without preventing RNA polymerase binding. However, contact between RNA polymerase and the -132 to -62 region of the non-coding strand of ndh DNA, and RNA polymerase-mediated open complex formation, were prevented by bound FNR. The upstream FNR-binding site (-94.5) was needed for efficient FNR-dependent repression of ndh transcription in vitro, and also for repression of an ndh-lacZ fusion in vivo. Anaerobic ndh repression may thus involve the binding of two pairs of FNR monomers upstream of the -35 region, which prevents essential RNA polymerase-DNA contacts in the upstream region as well as inhibiting RNA polymerase function by direct FNR interaction. Expression of the ndh-lacZ fusion in an fnr deletion strain was enhanced by anaerobic growth in rich medium or minimal medium supplemented with amino acids. Furthermore, two proteins (M(r) 12,000 and 35,000) which interact with and may activate transcription from the ndh promoter under these conditions were detected by gel retardation analysis. These putative amino acid-responsive activators may thus offset FNR-mediated repression and maintain a low level of anaerobic ndh expression for regulating the NAD+/NADH ratio during growth in rich media.
Summary FNR is a transcriptional regulator controlling the expression of a number of Escherichia coli genes in response to anoxia. It is structurally‐related to CRP (the cyclic AMP receptor protein) except for the presence of a cysteine‐rich N‐terminal extension, which may form part of an iron‐binding, redox‐sensing domain in FNR. Site‐directed substitution has previously shown that four of the cysteine residues (C20, C23, C29 and C122) are essential for FNR function, whereas the fifth (C16) is not. The FNR protein exists in two forms separable by non‐reducing SOS‐PAGE, and in studies with altered FNR proteins containing single substitutions at each of the five cysteine residues it was concluded that the faster‐migrating form (FNR(27)), possesses an intramolecular disulphide bond linking C122 to one of the cysteines near the N‐terminus. FNR(27)) was more abundant in aerobic cells but the physiological significance of this was not established. Footprint studies indicated that FNR proteins lacking essential cysteine residues are impaired in their ability to protect FNR sites in the ndh promoter. The non‐essential cysteine residue (C16) was identified as the source of the most reactive sulphydryl group and all of the inactive proteins exhibited different sulphydryl reactivities. The iron content of the C122A‐substituted protein was much reduced but those of the other proteins were less affected. Electrospray mass spectrometry confirmed the accuracy of the gene‐derived amino acid composition of FNR with a mutant protein and it showed that a fraction of the wild‐type protein may carry a 78 Da substituent which could not be removed with dithio‐threitol or β‐mercaptoethanol.
Two rapid and convenient methods have been developed for the amplification and purification of FNR, the anaerobic transcription regulator of Escherichia coli. The overproduced proteins resemble wild-type FNR in their basic properties: oligomeric state, iron contents (up to 2.7 atoms per monomer), DNA-binding affinities and ability to activate transcription. However, unlike previous preparations, FNR could be isolated in a form containing up to 0.25 atoms of acid-labile sulphur per monomer. Incorporation of iron increased the Mr of FNR from 28,000 to 40,000. Under anaerobic conditions, reconstituted FNR exhibited absorption maxima at 315 nm and 420 nm, which were replaced by a broad absorbance from 380 to 440 nm under aerobic conditions. These observations indicate that FNR contains one redox-sensitive [3Fe 4S] or [4Fe 4S] centre per monomer. Footprints of FNR-dependent promoters (ansB, fdn, fnr, narG, pflP6, pflP7 and nirB) showed protection at all of the predicted FNR sites except the pflP7 (-57.5), ansB (-74.5) and nirB (-89.5) sites. An unpredicted second binding site was detected at -57.5 in the narG promoter. Hypersensitive sites within regions of FNR protection indicated that FNR bends DNA in a similar way to CRP. Promoters containing binding sites for FNR (FF), CRP (CC) or hybrid sites (CF or FC) were footprinted with FNR and two derivatives (FNR-610 and FNR-573) which activate the CCmelR promoter in vivo. FNR preferentially protected the FNR site (FF) whereas FNR-610 preferred CC and FNR-573 interacted with equal affinity at all sites.
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