Properties of FNR proteins substituted at each of the five cysteine residues

Abstract

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.

HlyX, the FNR homologue of Actinobacillus pleuropneumoniae, is a [4Fe–4S]‐containing oxygen‐responsive transcription regulator that anaerobically activates FNR‐dependent Class I promoters via an enhanced AR1 contact

“…Green and J. R. Guest, unpublished) and in non-enzymically reconstituted FNR (Green et al, 1996b), and thus the 338 nm absorption does not arise as a consequence of using the GST-HlyX fusion. It is tempting to speculate that the single iron atom is associated with the equivalent of C122 of FNR (C121 in HlyX) which appears to be particularly important for iron binding in FNR (Green et al, 1993). It is probably safe to assume that the one-iron species is not of the rubredoxin type because such proteins have very distinctive visible spectra (Lovenburg, 1972).…”

FNR-dependent repression of ndh gene expression requires two upstream FNR-binding sites

“…3d). On its own, FNR" protected two regions of the ndh promoter (-61 to -40 and -105 to -84) corresponding to the FNR-I-and FNR-11-sites identified previously (Sharrocks et al, 1991;Green et al, 1993), and generated a hypersensitive site at an intermediate position, -73 (Fig. 3d).…”

“…1; Sharrocks et al, 1991;Green & Guest, 1994). The importance of FNR I1 was inferred from footprints of the ndh promoter with site-directed FNR variants which are unable to repress ndh expression in vivo (Sharrocks et al, 1990) but retain the capacity to protect FNR I but not FNR I1 (Green et al, 1993). The importance of FNR I1 was further emphasized by the reduction in ndh promoter activity (in vivo) and in FNR-mediated repression (in vitro) that accompanies the deletion of this upstream site (Green & Guest, 1994).…”

The molecular basis for the differential regulation of the hlyE-encoded haemolysin of Escherichia coli by FNR and HlyX lies in the improved Activating Region 1 contact of HlyX

“…All the members of this family are structurally related to CRP and, with the exception Downloaded from www.microbiologyresearch.org by IP: 54.191.190.102 On: Sat, 12 May 2018 16:52:15 J. G R E E N a n d M. L. BALDWIN HlyX also anaerobically activates the expression of a number of genes in E. coli K-12 strains, including a haemolysin, that FNR apparently does not (MacInnes et al, 1990;Green et al, 1992;Green & Baldwin, 1997).…”