Characterization of the nitric oxide-reactive transcriptional activator NorR.
The prokaryotic transcriptional regulator NorR is unusual in that it utilizes a mononuclear ferrous iron center rather than a heme moiety as a means of sensing nitric oxide (NO). Binding of NO to the nonheme iron center in the amino-terminal GAF domain of NorR results in formation of a mononitrosyl iron complex and relieves intramolecular repression within NorR, allowing this regulatory protein, a member of the sigma(54)-dependent family of enhancer-binding proteins, to activate expression of genes required for NO detoxification. This chapter describes detailed protocols for measuring transcriptional activation by Escherichia coli NorR in vivo and in vitro. It also details spectroscopic methods for analysis of the interaction of NO with the nonheme iron center and determination of the NO-binding affinity constant.
D'Autréaux
Benoît
B
Laboratoire Stress Oxydant et Cancer, Service de Biologie Intégrative et Génétique Moléculaire, Institut de Biologie et de Technologies de Saclay, CEA-Saclay, Gif-sur-Yvette Cedex, France.
Tucker
Nick
N
Spiro
Stephen
S
Dixon
Ray
R
D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D017736:Ion-Selective Electrodes / Q000592:standards; D007501:Iron / Q000737:chemistry; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D009573:Nitrites / Q000737:chemistry; D011485:Protein Binding; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*
D029968:Escherichia coli Proteins; D009573:Nitrites; C504602:NorR protein, E coli; D015534:Trans-Activators; D009569:Nitric Oxide; D007501:Iron