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Transcriptional co‐activation at the ansB promoters: involvement of the activating regions of CRP and FNR when bound in tandem

Previous work with semi‐synthetic promoters containing a single CRP binding site centred at 41.5 bp from the transcription start site has demonstrated enhanced transcription (synergism) when a second binding site, for CRP or FNR, is placed upstream at around −91 bp. The ansB promoter in Escherichia coli is co‐activated in a co‐dependent manner by one dimer each of CRP and FNR protein whose binding sites are at around −91 and −41 bp, respectively, from the transcription start site. Similarly, the homologous ansB promoter in Salmonella is co‐activated by two dimers of CRP which function synergistically. The binding sites at the E. coli promoter have been changed by mutation to provide a number of active promoter derivatives carrying other combinations of FNR and CRP binding sites. The co‐dependent versus synergistic interaction of these activators and their requirement for known activating regions have been examined. The results demonstrate that FNR can co‐activate when located upstream at around −91 bp in combination with either FNR or CRP downstream. When FNR occupies the downstream site the promoter is co‐dependent on an upstream activator, but not when CRP occupies this site. Activating region 1 in CRP (defined by substitutions at residue H159) and its putative equivalent in FNR (defined by substitutions at S73) are mainly required in the upstream activator; the putative equivalent in FNR of activating region 3 of CRP (defined by substitutions at G85 and K52, respectively) is mainly required in the dimer which binds downstream. Activating region 1 of FNR is required only in the downstream subunit of the upstream activator in a promoter which is co‐dependent on two FNR dimers. These data suggest that both bound upstream and downstream activators interact with RNA polymerase to promote transcription, and that co‐dependence is determined by the nature of the activator plus the promoter context.