In the archetypal bacterial transcription, the multi-subunit core RNA polymerase (RNAP) is
specifically bound to the promoter through the sigma factor (σ), forming a closed complex
where DNA remains double-stranded. The promoter DNA is initially spontaneously melted
by the σ factor within the core enzyme and subsequently loaded into the active channel of
the holoenzyme. However, a major alternative transcription paradigm that depends on the
sigma factor σ54 also exists in bacteria and controls pathogenicity, biofilm formation,
bioluminescence, nitrogen fixation and stress responses. σ54 forms inhibitory interactions
with DNA in the closed complex, which can only be alleviated by ATP hydrolysis-driven
remodelling catalysed by bacterial enhancer binding proteins (bEBPs). In this regard, σ54-
dependent transcription can be viewed analogous to the eukaryotic RNA Pol II system. This
study was initiated to advance the understanding of: (i) how the ATP hydrolysis energy is
relayed from the ATPase catalytic site to the closed complex for remodelling, (ii) the
hexameric interface organisation of bEBPs for ATP hydrolysis, and (iii) the role of core RNAP
in σ54-dependent transcription. A newly devised cross-linking technique combined with the
DNA footprinting methods provided new insights of the organisation of each transcription
component. The data gathered from this study updated the current working model for ATPdependent
transcription. In addition, the cross-linking method proved to be an excellent
tool to study protein-protein and nucleo-protein interactions.