Regulatory sequences in sigma 54 localise near the start of DNA melting

Transcription initiation by the enhancer-dependent σ54 RNA polymerase holoenzyme is positively regulated after promoter binding. The promoter DNA melting process is subject to activation by an enhancer-bound activator protein with nucleoside triphosphate hydrolysis activity. Tethered iron chelate probes attached to amino and carboxyl-terminal domains of σ54 were used to map σ54-DNA interaction sites. The two domains localise to form a centre over the −12 promoter region. The use of deletion mutants of σ54 suggests that amino-terminal and carboxyl-terminal sequences are both needed for the centre to function. Upon activation, the relationship between the centre and promoter DNA changes. We suggest that the activator re-organises the centre to favour stable open complex formation through adjustments in σ54-DNA contact and σ54 conformation. The centre is close to the active site of the RNA polymerase and includes σ54 regulatory sequences needed for DNA melting upon activation. This contrasts systems where activators recruit RNA polymerase to promoter DNA, and the protein and DNA determinants required for activation localise away from promoter sequences closely associated with the start of DNA melting.