Activity of purified NIFA, a transcriptional activator of nitrogen fixation genes.
The NIFA protein activates transcription of nitrogen fixation (nif) operons by the sigma 54-holoenzyme form of RNA polymerase. We purified active NIFA from Klebsiella pneumoniae in the form of a maltose-binding protein (MBP)-NIFA fusion; proteolytic release of MBP yielded inactive and insoluble NIFA. MBP-NIFA activated transcription from the nifHDK promoter in a purified transcription system. Like the related transcriptional activator NTRC, MBP-NIFA catalyzed the ATP-dependent isomerization of closed complexes between sigma 54-holoenzyme and a promoter to open complexes. MBP-NIFA had a broader nucleotide specificity than NTRC, being able to utilize pyrimidine in addition to purine nucleoside triphosphates. Both MBP-NIFA and a purified C-terminal fragment of NIFA bound to the upstream activation sequence for the nifHDK promoter, as assessed by DNAse I footprinting. When assays were performed at 37 degrees C instead of the usual 30 degrees C, transcriptional activation, open complex formation, and DNA binding by MBP-NIFA were all abolished, consistent with the known heat lability of NIFA. However, the purified C-terminal fragment of NIFA still bound the upstream activation sequence at 37 degrees C, indicating that the function of the helix-turn-helix DNA-binding motif is not inherently heat-labile.
Lee
H S
HS
Department of Plant Pathology, University of California, Berkeley 94720.
Berger
D K
DK
Kustu
S
S
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009586:Nitrogen Fixation / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D009876:Operon; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
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; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C461599:maltose transport system, E coli