Trapping of a Transcription Complex Using a New Nucleotide Analogue: AMP Aluminium Fluoride

Mechanochemical proteins rely on ATP hydrolysis to establish the different functional states required for their biological output. Studying the transient functional intermediate states these proteins adopt as they progress through the ATP hydrolysis cycle is key to understanding the molecular basis of their mechanism. Many of these intermediates have been successfully ‘trapped’ and functionally characterised using ATP analogues. Here, we present a new nucleotide analogue, AMP–AlFx, which traps PspF, a bacterial enhancer binding protein, in a stable complex with the σ54-RNA polymerase holoenzyme. The crystal structure of AMP–AlFx•PspF1–275 provides new information on protein–nucleotide interactions and suggests that the β and γ phosphates are more important than the α phosphate in terms of sensing nucleotide bound states. In addition, functional data obtained with AMP–AlFx establish distinct roles for the conserved catalytic AAA+ (ATPases associated with various cellular activities) residues, suggesting that AMP–AlFx is a powerful new tool to study AAA+ protein family members and, more generally, Walker motif ATPases.