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-AlF(x), which traps PspF, a bacterial enhancer binding protein, in a stable complex with the sigma(54)-RNA polymerase holoenzyme. The crystal structure of AMP-AlF(x)*PspF(1-275) provides new information on protein-nucleotide interactions and suggests that the beta and gamma phosphates are more important than the alpha phosphate in terms of sensing nucleotide bound states. In addition, functional data obtained with AMP-AlF(x) establish distinct roles for the conserved catalytic AAA(+) (ATPases associated with various cellular activities) residues, suggesting that AMP-AlF(x) is a powerful new tool to study AAA(+) protein family members and, more generally, Walker motif ATPases.
Joly
Nicolas
N
Division of Biology, Sir Alexander Fleming Building, Imperial College London, Exhibition Road, South Kensington, London SW7 2AZ, UK.
Rappas
Mathieu
M
Buck
Martin
M
Zhang
Xiaodong
X
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D017607:Aluminum Compounds / Q000378:metabolism*; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005459:Fluorides / Q000378:metabolism*; D015636:Magnesium Chloride / Q000378:metabolism; D008956:Models, Chemical; D009711:Nucleotides / Q000378:metabolism*; D009942:Organometallic Compounds / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54 / Q000378:metabolism; D016962:Sinorhizobium meliloti / Q000235:genetics; D013059:Spectrum Analysis, Raman; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014961:X-Ray Diffraction
D017607:Aluminum Compounds; D029968:Escherichia coli Proteins; D009711:Nucleotides; D009942:Organometallic Compounds; C098954:PspF protein, E coli; D015534:Trans-Activators; D015636:Magnesium Chloride; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D051877:RNA Polymerase Sigma 54; D000251:Adenosine Triphosphatases; D005459:Fluorides; C032311:aluminum fluoride