Comparative analysis of activator-Eσ(54) complexes formed with nucleotide-metal fluoride analogues

Bacterial RNA polymerase (RNAP) containing the major variant σ(54) factor forms open promoter complexes in a reaction in which specialized activator proteins hydrolyse ATP. Here we probe binding interactions between σ(54)-RNAP (Eσ(54)) and the ATPases associated with various cellular activities (AAA+) domain of the Escherichia coli activator protein, PspF, using nucleotide-metal fluoride (BeF and AlF) analogues representing ground and transition states of ATP, which allow complexes (that are otherwise too transient with ATP) to be captured. We show that the organization and functionality of the ADP–BeF- and ADP–AlF-dependent complexes greatly overlap. Our data support an activation pathway in which the initial ATP-dependent binding of the activator to the Eσ(54) closed complex results in the re-organization of Eσ(54) with respect to the transcription start-site. However, the nucleotide-dependent binding interactions between the activator and the Eσ(54) closed complex are in themselves insufficient for forming open promoter complexes when linear double-stranded DNA is present in the initial closed complex.