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

Abstract

The ndh gene of Escherichia coli encodes a non-proton-translocating NADH dehydrogenase (Ndhll) that is anaerobically repressed by the global transcription regulator, FNR. FNR binds at two sites (centred at -505 and University of Sheff ield,-Firth Court, Western Bank, Sheffield 510 ZTN, UK -94.5) in the ndh promoter but the mechanism of FNR-mediated repression appears not to be due to promoter occlusion. This mechanism has been investigated using an aerobically active derivative of FNR, FNR* (FNR-D154A), with ndh promoters containing altered FNR-binding sites. FNR* repressed ndh gene expression both aerobically and anaerobically in wiwo. Gel retardation analysis and DNase I footprinting with purified FNR* protein confirmed that FNR interacts at two sites in the ndh promoter, and that FNR and RNA polymerase (RNAP) can bind simultaneously. Studies with three altered ndh promoters, each containing an impaired or improved FNR-site, indicated that both FNR-sites are needed for efficient repression in wiwo. The a-subunit of RNAP interacted with two regions (centred at -105 and -46), each overlapping one of the FNR-sites in the ndh promoter. Footprints of the FNR*-RNAP-ndh ternary complex indicated that FNR*-binding at -505 prevents the a-subunit of RNAP from docking with the DNA just upstream of the -35 element. Binding of a second FNR* molecule at the -105 site likewise prevents binding of the a-subunit at its alternative site, thus providing a plausible mechanism for FNR-mediated repression based on displacement of the a-subunit of RNAP.

Downregulation of Escherichia coli yfiD expression by FNR occupying a site at −93.5 involves the AR1‐containing face of FNR

“…Thus, in an appropriate context FNR can activate transcription when positioned at ¹41 (FFpmelR ), and additional upstream FNR sites can produce inhibitory ( yfiD þ FNR), neutral/positive ( yfiD þ FNR variants or HlyX) or activating (FF-FFansB þ FNR) interactions. The potential for switching between activation and repression of transcription merely by altering the position of an upstream FNR site suggests that FNR repressible promoters, such as that of the ndh gene (Meng et al, 1997), may have evolved through duplication and shuffling of the positions of FNR sites. By this mechanism, promoter regions that display the full range of FNR regulation, from synergistic activation to full repression, could be generated.…”

“…In conclusion, the yfiD promoter joins the ndh (Meng et al, 1997) and fdnGHI (Guest et al, 1996) promoters as an FNR-regulated promoter with an inhibitory upstream FNR site. The inhibitory action of FNR bound at the most upstream site appears to be mediated through proteinprotein contacts involving the face of FNR that includes AR1.…”

In vivo cycling of the Escherichia coli transcription factor FNR between active and inactive states

“…Measurement of the relative amounts of an FNR-activated transcript (dmsA; Lamberg & Kiley, 2000) and an FNR-repressed transcript (ndh; Green & Guest, 1994;Meng et al, 1997;Spiro et al, 1989) in anaerobic cultures of MG1655 and MG1655 clpX revealed that the dmsA transcript was~2?5-fold more abundant, and the ndh transcript was~1?5-fold less abundant in the clpX mutant (Fig. 2b).…”

“…The [4Fe-4S] form of FNR acts as both a positive and a negative regulator of gene expression, activating transcription by recruiting RNA polymerase, or repressing transcription by inhibiting the formation of productive promoter-RNA polymerase interactions (Barnard et al, 2004;Bell & Busby, 1994;Blake et al, 2003;Browning et al, 2003;Green & Marshall, 1999;Green et al, 1998;Lamberg & Kiley, 2000;Lamberg et al, 2002;Li et al, 1998; Lonetto et al, 1998;Marshall et al, 2001;Meng et al, 1997;Williams et al, 1997;Wing et al, 2000). Exposure of E. coli to air is sensed by the , 2005).…”

“…Accordingly, deletion of the iscS gene (a cysteine desulphurase that provides the sulphur for iron-sulphur cluster assembly) impairs FNR activity in vivo (Schwartz et al, 2000). Furthermore, comparison of the effects on FNR, which requires an iron-sulphur cluster for activity, and those on FNR*, an FNR variant that is active in the absence of an iron-sulphur cluster, suggests that the isc operon plays a major role in the assembly of the FNR ironsulphur cluster (Schwartz et al, 2000).The [4Fe-4S] form of FNR acts as both a positive and a negative regulator of gene expression, activating transcription by recruiting RNA polymerase, or repressing transcription by inhibiting the formation of productive promoter-RNA polymerase interactions (Barnard et al, 2004;Bell & Busby, 1994;Blake et al, 2003;Browning et al, 2003;Green & Marshall, 1999;Green et al, 1998;Lamberg & Kiley, 2000;Lamberg et al, 2002;Li et al, 1998; Lonetto et al, 1998;Marshall et al, 2001;Meng et al, 1997;Williams et al, 1997;Wing et al, 2000). Exposure of E. coli to air is sensed by the , 2005).…”