Interactions of regulated and deregulated forms of the σ(54) holoenzyme with heteroduplex promoter DNA

The bacterial σ(54) RNA polymerase holoenzyme binds to promoters as a stable closed complex that is silent for transcription unless acted upon by an enhancer-bound activator protein. Using DNA binding and transcription assays the ability of the enhancer-dependent σ(54) holoenzyme to interact with promoter DNA containing various regions of heteroduplex from –12 to –1 was assessed. Different DNA regions important for stabilising σ(54) holoenzyme–promoter interactions, destabilising binding, limiting template utilisation in activator-dependent transcription and for stable binding of a deregulated form of the holoenzyme lacking σ(54) Region I were identified. It appears that homoduplex structures are required for early events in σ(54) holoenzyme promoter binding and that disruption of a repressive fork junction structure only modestly deregulates transcription. DNA opening from –5 to –1 appears important for stable engagement of the holoenzyme following activation. The regulatory Region I of σ(54) was shown to be involved in interactions with the sequences in the –5 to –1 area.