NarL‐phosphate must bind to multiple upstream sites to activate transcription the narG promoter of Escherichia coli

Abstract

The stimulation of Fnr-dependent transcription from the narG promoter by NarL-phosphate is known to require a cis-acting sequence, the NarL box, located approximately 195 bp upstream from the transcription start site, and the interaction of integration host factor (IHF) with a binding site in the intervening region (positions -110 to -140) between the NarL box and the transcription start site. By gel retardation and DNase I protection studies, we have demonstrated that NarL-phosphate, produced by the reaction of purified NarL with acetyl phosphate, specifically binds to a fragment derived from the upstream region of the narG promoter. The fragment was protected by NarL-phosphate binding to two distinct regions. One was an extended sequence of approximately 40 bp surrounding the NarL box at -195; the second was located downstream from the IHF-binding region and included a sequence extending from positions -80 to -120. Alteration by site-directed mutagenesis of a putative inverted NarL box sequence identified within the downstream protected region in a plasmid containing a narG-lacZ fusion eliminated the NarL-phosphate-mediated stimulation of transcription. NarL-phosphate bound to the two regions independently from IHF binding and it bound to each site independently when the two sites were separated by cleavage of the promoter fragment. Stimulation of transcription from the narG promoter by NarL-phosphate appears to result from the formation of a folded protein-DNA structure created by the binding of NarL-phosphate to multiple sites on either side of an IHF-induced bend in the upstream region of the promoter.

Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K‐12 depends on DNA binding site arrangement

“…Half-maximal protection of the nirB 7-2-7 heptamer pair occurred with less than 0.4 M MBP-NarL monomers and less than 3 M MBP-NarP monomers. This heptamer pair is therefore a much higher affinity NarLbinding site than those previously characterized (data not shown; Li et al, 1994;Walker and DeMoss, 1994;Kaiser and Sawers, 1995;Darwin and Stewart, 1995). This is consistent with the observation that full NarL-dependent induction of nirB operon expression occurs in the presence of nitrite, when the concentration of phospho-NarL is thought to be relatively low.…”

“…Results of DNase I footprinting experiments corroborate the identification of NarL heptamers in the narG, fdnG (formate dehydrogenase-N), narK (nitrite extrusion protein), frdA, pfl (pyruvate-formate lyase) and napF (periplasmic nitrate reductase) control regions (Li et al, 1994;Walker and DeMoss, 1994;Darwin and Stewart, 1995;Kaiser and Sawers, 1995). Many (but not all) NarL-binding sites are arranged as an inverted pair of heptamers with 2 bp spacing.…”

“…We do not fully understand the regulation of narG operon expression by the NarL protein. However, it is clear that the binding of phospho-NarL to these multiple single heptamers is a cooperative process (Li et al, 1994;Walker and DeMoss, 1994;. We were unable to obtain a DNase I footprint of the narG operon control region with phosphorylated MBP-NarP protein (not shown).…”

Molecular Characterization of the PmrA Regulon

“…However, like the PhoP protein of Bacillus subtilis, the PmrA protein dimerizes and binds to DNA regardless of its phosphorylation state (28). Several response regulators also bind to their target promoters efficiently in their unphosphorylated form (29,30), but others, such as NarL and ComA, bind to their target genes only in phosphorylated form (31,32). Yet, in all cases, phosphorylation of response regulators affects binding to the target promoters, often by increasing binding affinity such as in the case of the OmpR and PhoB proteins (33,34).…”

“…Phosphorylation of PmrA by PmrBc-The PmrBc-H6 or PmrA-H6 proteins were incubated at room temperature (RT) for 10 or 40 min, respectively, with 50 Ci of [␥- 32 DNase I Footprinting-DNase I protection assays were done for both DNA strands using the appropriate labeled primer. PmrA-H6 protein was incubated with phosphorylated or unphosphorylated PmrBc-H6 for 20 min at RT as described above, except that instead of [␥-…”