Trapping of a transcription complex using a new nucleotide analogue: AMP aluminium fluoride

JOURNAL OF MOLECULAR BIOLOGY

卷:375

Article

Joly, Nicolas¹  Rappas, Mathieu²  Buck, Martin¹  Zhang, Xiaodong² 

[1] Univ London Imperial Coll Sci Technol & Med, Div Biol, London SW7 2AZ, England

[2] Univ London Imperial Coll Sci Technol & Med, Div Mol Biosci, London SW7 2AZ, England

关键词: AAA(+) ATPase;    nucleotide analogue;    transcription;    bEBP;    PspF;   

DOI  :  10.1016/j.jmb.2007.11.050

来源: Elsevier

【 摘 要 】

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-AIF(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-AIF(x)circle PspF(1-275) provides new information on protein-nucleotide interactions and suggests that the and gamma phosphates are more important than the a phosphate in terms of sensing nucleotide bound states. In addition, functional data obtained with AMP-AIF(x) establish distinct roles for the conserved catalytic AAA(+) (ATPases associated with various cellular activities) residues, suggesting that AMP-AIF(x) is a powerful new tool to study AAA(+) protein family members and, more generally, Walker motif ATPases. (C) 2007 Elsevier Ltd. All rights reserved.

【 授权许可】

Free