Structural mechanism of sigma54‐dependent AAA+ ATPases

Bacteria use σ54‐dependent transcription to regulate genes to survive, promote diseases and traverse complex developmental pathways. Eσ54 RNAP is unable to start transcription without assistance from activators called enhancer binding proteins (EBPs), such as NtrC1 from Aquifex aeolicus . An EBP uses the energy of ATP binding and hydrolysis to enable Eσ54 to start transcription. EBPs like NtrC1 are AAA+ ATPases and function as homo‐oligomeric, ring‐shaped assemblies with active sites located at each intersubunit interface. We recently proposed a model in which structural changes induced by a single nucleotide binding event can propagate throughout the ring. To analyze changes induced by consecutive nucleotide binding we measured time‐resolved small‐angle X‐ray scattering. The results showed that upon nucleotide binding NtrC1 undergoes a series of large scale conformational changes with at least one intermediate and clearly separated fast and slow phases. This knowledge enabled crystallization of a new oligomeric state ‐ an open, asymmetric, hexameric ring of apo, ADP‐bound and ADP‐BeF x ‐bound subunits. Position within the opened ring correlated with a subunit's affinity for ATP. We suggest that sparse occupancy integrated with cooperative loop extension and retraction facilitates initial binding to Eσ54 and its subsequent manipulation to promote the transition from closed to opened promoter complex.