In vitro activity of NifL, a signal transduction protein for biological nitrogen fixation.
In the free-living diazotroph Klebsiella pneumoniae, the NifA protein is required for transcription of all nif (nitrogen fixation) operons except the regulatory nifLA operon itself. NifA activates transcription of nif operons by the alternative holoenzyme form of RNA polymerase, sigma 54 holoenzyme. In vivo, NifL is known to antagonize the action of NifA in the presence of molecular oxygen or combined nitrogen. We now demonstrate inhibition by NifL in vitro in both a coupled transcription-translation system and a purified transcription system. Crude cell extracts containing NifL inhibit NifA activity in the coupled system, as does NifL that has been solubilized with urea and allowed to refold. Inhibition is specific to NifA in that it does not affect activation by NtrC, a transcriptional activator homologous to NifA, or transcription by sigma 70 holoenzyme. Renatured NifL also inhibits transcriptional activation by a maltose-binding protein fusion to NifA in a purified transcription system, indicating that no protein factor other than NifL is required. Since inhibition in the purified system persists anaerobically, our NifL preparation does not sense molecular oxygen directly.
Lee
H S
HS
Department of Plant Biology, University of California, Berkeley 94720.
Narberhaus
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F
Kustu
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D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000187:drug effects* / Q000494:pharmacology*; D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000235:genetics*; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009586:Nitrogen Fixation / Q000235:genetics*; D014176:Protein Biosynthesis / Q000187:drug effects; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D014157:Transcription Factors / Q000187:drug effects*; D014158:Transcription, Genetic / Q000187:drug effects
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; C061338:NifA protein, Bacteria; D011994:Recombinant Proteins; D014157:Transcription Factors; C461599:maltose transport system, E coli; C084296:nifL protein, Bacteria