TRANSCRIPTIONAL COACTIVATION 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.