DNA melting within a binary σ54-promoter DNA complex

Abstract

The σ54 subunit of the bacterial RNA polymerase requires the action of specialized enhancer-binding activators to initiate transcription. Here we show that σ54 is able to melt promoter DNA when it is bound to a DNA structure representing the initial nucleation of DNA opening found in closed complexes. Melting occurs in response to activator in a nucleotide-hydrolyzing reaction and appears to spread downstream from the nucleation point toward the transcription start site. We show that σ54 contains some weak determinants for DNA melting that are masked by the Region I sequences and some strong ones that require Region I. It seems that σ54 binds to DNA in a self-inhibited state, and one function of the activator is therefore to promote a conformational change in σ54 to reveal its DNA-melting activity. Results with the holoenzyme bound to early melted DNA suggest an ordered series of events in which changes in core to σ54 interactions and σ54-DNA interactions occur in response to activator to allow σ54 isomerization and the holoenzyme to progress from the closed complex to the open complex.