Expression, purification, and functional analysis of the C-terminal domain of Herbaspirillum seropedicae NifA protein.
The Herbaspirillum seropedicae NifA protein is responsible for nif gene expression. The C-terminal domain of the H. seropedicae NifA protein, fused to a His-Tag sequence (His-Tag-C-terminal), was over-expressed and purified by metal-affinity chromatography to yield a highly purified and active protein. Band-shift assays showed that the NifA His-Tag-C-terminal bound specifically to the H. seropedicae nifB promoter region in vitro. In vivo analysis showed that this protein inhibited the Central + C-terminal domains of NifA protein from activating the nifH promoter of K. pneumoniae in Escherichia coli, indicating that the protein must be bound to the NifA-binding site (UAS site) at the nifH promoter region to activate transcription.
Monteiro
Rose A
RA
Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná CP 19046, Curitiba, PR 81531-990, Brazil.
Souza
Emanuel M
EM
Geoffrey Yates
M
M
Steffens
M Berenice R
MB
Pedrosa
Fábio O
FO
Chubatsu
Leda S
LS
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000502:physiology; D020562:Betaproteobacteria / Q000378:metabolism*; D002845:Chromatography; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification* / Q000502:physiology; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D001426:Bacterial Proteins; C061338:NifA protein, Bacteria; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA