Communication between Eσ54, promoter DNA and the conserved threonine residue in the GAFTGA motif of the PspF σ54‐dependent activator during transcription activation

Summary Conversion of Eσ54 closed promoter complexes to open promoter complexes requires specialized activators which are members of the AAA (ATPases Associated with various cellular Activities) protein family. The ATP binding and hydrolysis activity of Eσ54 activators is used in an energy coupling reaction to remodel the Eσ54 closed promoter complex and to overcome the σ54‐imposed block on open complex formation. The remodelling target for the AAA activator within the Eσ54 closed complex includes a complex interface contributed to by Region I of σ54, core RNA polymerase and a promoter DNA fork junction structure, comprising the Eσ54 regulatory centre. One σ54 binding surface on Eσ54 activators is a conserved sequence known as the GAFTGA motif. Here, we present a detailed characterization of the interaction between Region I of σ54 and the Escherichia coli AAA σ54 activator Phage shock protein F. Using Eσ54 promoter complexes that mimic different conformations adopted by the DNA during open complex formation, we investigated the contribution of the conserved threonine residue in the GAFTGA motif to transcription activation. Our results suggest that the organization of the Eσ54 regulatory centre, and in particular the conformation adopted by the σ54 Region I and the DNA fork junction structure during open complex formation, is communicated to the AAA activator via the conserved T residue of the GAFTGA motif.