Interactions of regulated and deregulated forms of the σ54 holoenzyme with heteroduplex promoter DNA

Abstract

The bacterial σ54 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 σ54 holoenzyme to interact with promoter DNA containing various regions of heteroduplex from -12 to -1 was assessed. Different DNA regions important for stabilising σ54 holoenzyme-promoter interactions, destabilising binding, limiting template utilisation in activator-dependent transcription and for stable binding of a deregulated form of the holoenzyme lacking σ54 Region I were identified. It appears that homoduplex structures are required for early events in σ54 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 σ54 was shown to be involved in interactions with the sequences in the -5 to -1 area.