Functional studies of the ‘GAFTGA’ motif of Escherichia coli Phage Shock Protein F

Abstract

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.