Regulation of transcription at the ndh promoter of Escherichia coli by FNR and novel factors

Abstract

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.

Aerobic regulation of cytochrome d oxidase (cydAB ) operon expression in Escherichia coli: roles of Fnr and ArcA in repression and activation

“…3). The location of the upstream Fnr site 1, separated by 18 bp from the ¹35 region, is atypical of the locations of Fnr binding sites for other Fnr repressed genes including ndh and fnr (Green and Guest, 1994 Fnr-dependent regulation of cydAB expression will require additional in vitro and in vivo experiments involving sitedirected mutagenesis of each Fnr sequence to evaluate their contribution in regulating cydAB transcription. Finally, the earlier proposals that Fnr does not act directly to regulate cydAB gene expression must now be re-evaluated (Gennis and Stewart, 1996;Lynch and Lin, 1996a).…”

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

“…la). This probably explains why the ndh-lac2 translational fusions of two plasmids (pGS602 and pGS603) derived from pGS418 had very low activities (Green & Guest, 1994). Microbiological methods.…”

“…2a) by PCR amplification of an fnr" fragment and reconstruction of the intact and resequenced fnr* gene in a 1156 bp NcoI-Hind111 fragment that was subsequently cloned in pGEX-KG. The primary source of ndh promoter DNA was pGS418 (Green & Guest, 1994). A derivative of PALTER-E X I , pGS828, containing the 430 bp EcoRI-BamHI, ndh promoter (Pndh) fragment from pGS418, was used to create three altered ndh promoters (Fig.…”

“…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). Distant upstream FNR-sites have also been shown to be important for efficient repression of the fnr and narX promoters (Takahashi et al, 1994).…”

“…GREEN a n d J. R. GUEST TTGAT----ATCAA) which is centred at -50.5 and contains a perfect match to the FNR-site consensus, and a low-affinity site (FNR 11; TTGAT----ACCCG) which is centred further upstream at -94-5 and matches the consensus in only one half-site ( Fig. 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).…”