Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL.
The prokaryotic enhancer-binding protein NifA stimulates transcription at a distance by binding to sequences upstream of nitrogen fixation (nif) promoters and catalyzing the formation of open promoter complexes by RNA polymerase containing the alternative sigma factor, sigma 54. The activity of NifA in vivo is modulated by the negative regulatory protein NifL in response to environmental oxygen and fixed nitrogen. To date, a detailed biochemical analysis of these proteins from the model diazotroph Klebsiella pneumoniae has been hindered by their insolubility. We have now purified NifA and NifL from Azotobacter vinelandii in their native form. NifA is competent in specific DNA binding, transcriptional activation, and response to negative regulation by NifL in vitro. In contrast to the conserved mechanism of phosphotransfer demonstrated by other two-component regulatory systems, our results support a model in which NifL regulates the activity of NifA via a protein-protein steric block interaction rather than a catalytic modification of NifA.
Austin
S
S
Nitrogen Fixation Laboratory, University of Sussex, Brighton, England.
Buck
M
M
Cannon
W
W
Eydmann
T
T
Dixon
R
R
D016948:Azotobacter vinelandii / Q000235:genetics*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000235:genetics; D009586:Nitrogen Fixation / Q000235:genetics*; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C061338:NifA protein, Bacteria; D009711:Nucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors; C084296:nifL protein, Bacteria