The multiple roles of CRP at the complex acs promoter depend on activation region 2 and IHF.

acs encodes a high-affinity enzyme that permits survival during carbon starvation. As befits a survival gene, its transcription is subject to complex regulation. Previously, we reported that cAMP receptor protein (CRP) activates acs transcription by binding tandem DNA sites located upstream of the major acsP2 promoter and that the nucleoid protein IHF (integration host factor) binds three specific sites located just upstream. In vivo, the sequence that includes these IHF sites exerts a positive effect on CRP-dependent transcription, while a construct containing only the most proximal site exhibits reduced transcription compared with the full-length promoter or with a construct lacking all three IHF sites. Here, we defined the minimal system required for this IHF-dependent inhibition, showing it requires the promoter-distal CRP site and an amino acid residue located within activation region 2 (AR2), a surface determinant of CRP that interacts with RNA polymerase (RNAP). Surprisingly, for a Class III promoter, disruption of AR2 caused significant changes in the activity and structure of both the full-length promoter and the construct with the single proximal IHF site. We propose that AR2, together with IHF, mediates formation of a multi-protein complex, in which RNAP is stabilized in an open complex that remains poised on the promoter ready to respond rapidly to environmental changes.

Integration of three signals at the Escherichia coli nrf promoter: a role for Fis protein in catabolite repression.

Expression from the Escherichia coli nrf operon promoter is activated by the anaerobically triggered transcription factor, FNR, and by the nitrate/nitrite ion-controlled response regulators, NarL or NarP, but is repressed by the IHF and Fis proteins. Here, we present in vitro studies on the nrf promoter, using permanganate footprinting to measure open complex formation, and DNase I footprinting to monitor binding of the different regulators and the interactions between them. Our results show that open complex formation is completely dependent on FNR and is enhanced by NarL, but is repressed by IHF or Fis. NarL counteracts repression by IHF but is unable to alter repression by Fis. These results suggest mechanisms by which nrf promoter activity is modulated by the different factors. Expression from the nrf promoter is known to be repressed in rich media, especially in the presence of glucose, but the molecular basis of this is not understood. Here, we show that this catabolite repression is relieved by mutations that weaken the DNA site for Fis, improve the DNA site for FNR or improve the promoter -10 or -35 elements. Hence, Fis protein is a major factor responsible for catabolite repression at the nrf promoter, and Fis can override activation by FNR and NarL or NarP.

Modulation of CRP-dependent transcription at the Escherichia coli acsP2 promoter by nucleoprotein complexes: anti-activation by the nucleoid proteins FIS and IHF.

acs encodes acetyl-coenzyme A synthetase, a high-affinity enzyme that allows cells to scavenge for acetate during carbon starvation. CRP activates acs transcription by binding tandem DNA sites located upstream of the major promoter, acsP2. Here, we used electrophoretic mobility shift assays and DNase I footprint analyses to demonstrate that the nucleoid proteins FIS and IHF each bind multiple sites within the acs regulatory region, that FIS competes successfully with CRP for binding to their overlapping and neighbouring sites and that IHF binds independently of either FIS or CRP. Using in vitro transcription assays, we demonstrated that FIS and IHF independently reduce CRP-dependent acs transcription. Using in vivo reporter assays, we showed that disruption of DNA sites for FIS or deletion of DNA sites for IHF increases acs transcription. We propose that FIS and IHF each function directly as anti-activators of CRP, each working independently at different times during growth to set the levels of CRP-dependent acs transcription.

Cyclic AMP receptor protein-dependent activation of the Escherichia coli acsP2 promoter by a synergistic class III mechanism.

The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by "focusing" RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions -69.5 and -122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the alpha subunit of RNA polymerase holoenzyme (alpha-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the alpha-CTD. Other surface-exposed residues in the alpha-CTD contributed to acs transcription, suggesting that the alpha-CTD may interact with at least one protein other than CRP.

Independent regulation of the divergent Escherichia coli nrfA and acsP1 promoters by a nucleoprotein assembly at a shared regulatory region.

Expression from the Escherichia coli nrfA promoter (pnrfA) is activated by both the FNR protein (an anaerobically triggered transcription activator) and the NarL or NarP proteins (transcription activators triggered by nitrite and nitrate). Under anaerobic conditions, FNR binds to a site centred at position -41.5 at pnrfA and activates transcription. Further activation, induced by the presence of nitrite, results from the binding of NarL and NarP to a site centred at position -74.5. A second promoter (pacsP1), which directs transcription into the adjacent gene encoding acetyl coenzyme A synthetase (acs), is overlapping and divergent to pnrfA. Despite extensive overlap of regulatory elements, pnrfA and pacsP1 are regulated independently. We demonstrate that at least two nucleoid-associated factors bind to the nrfA-acs intergenic region. The Fis protein binds to a site centred at position -15 (in relation to pnrfA transcription), whereas the IHF protein binds to a site centred at position -54. Both Fis and IHF repress in vivo expression from pacsP1, but have smaller repressive effects on expression from pnrfA. Gel retardation assays were used to investigate the pairwise binding of FNR, NarL, Fis and IHF proteins to the nrfA-acs intergenic region. The binding of NarL and IHF is mutually exclusive, whereas all other combinations can bind simultaneously. Experiments in which deletions and point mutations were introduced into the upstream region of pnrfA demonstrated that an additional factor must bind upstream to inhibit FNR-dependent transcription. We conclude that the nrfA-acs intergenic region is folded into an ordered nucleoprotein structure that permits the two divergent promoters to be regulated independently in response to different physiological signals.