Bacterial transcription initiation relies on the binding of a dissociable sigma factor to the catalytic RNA polymerase (RNAP) ‘core’ enzyme to enable promoter specific DNA recognition. While the initial σ70 RNAP-promoter DNA bound complex (termed the closed complex) can spontaneously isomerise to form open promoter complexes, RNAP containing the major variant, the σ54 factor requires activation by bacterial enhancer binding proteins (bEBPs) which reorganize the initial closed promoter complex to an open complex in an ATP consuming reaction. These bEBPs belong to the AAA+ (ATPases associated with various cellular activities) superfamily of proteins that form oligomeric rings (often hexamers) on upstream activation sequences (UAS) ~150bp upstream of the promoter. A DNA looping event, often mediated by integration host factor (IHF), brings the bEBP in close proximity to the σ54-RNAP closed complex (RPc). The interaction between σ54 (within the context of the RPc) and the bEBP is strictly dependent on a sequence insertion (that is common to all bEBPs) termed the L1 loop, which is thought to be the major structural determinant in energy transfer between the bEBP and σ54. The σ54 subunit is split up into three distinct regions (termed Regions I, II and III), where the L1 loop interacts with the mobile Region I and Region III forms the DNA contacts necessary for engaging with -12 and -24 consensus sequences on promoter DNA.
A basic understanding of the global interactions that mediate σ54-dependent transcription has already been obtained, yet many specific mechanistic details remain unknown. In this study we elucidate three features of σ54 transcription activation: i) The interactions required within the bEBP complex for functional oligomerisation, ii) the mechanism that transcription activation determinants act and iii) the role of Region III in binding to the -12 element.