In vitro studies of the domains of the nitrogen fixation regulatory protein NIFA.
The prokaryotic enhancer-binding protein NIFA is a multidomain transcriptional activator that catalyzes the formation of open complexes at nitrogen fixation (nif) promoters by a specialized form of RNA polymerase containing sigma 54. The NIFA protein from Klebsiella pneumoniae consists of three domains: the N-terminal domain of unknown function; the central catalytic domain, which is sufficient for transcriptional activation; and the C-terminal DNA-binding domain. Purified fusion proteins between maltose-binding protein (MBP) and NIFA deleted of its N-terminal domain (MBP-delta N-NIFA) or its C-terminal domain (MBP-NIFA-delta C) activated transcription from the K. pneumoniae nifH promoter both in vitro and in vivo. We previously showed that the same was true for a fusion between MBP and the central domain of NIFA. These results indicate that NIFA is sufficiently modular for all fusions carrying its catalytic domain to be active. Unexpectedly, however, simple predictions regarding the location of determinants of the heat lability and insolubility of NIFA, which were based on previous studies of its isolated central and C-terminal domains, were not borne out. Contrary to a previous report from this laboratory, we found that the in vitro start site of transcription for the K. pneumoniae nifH operon could be either of two adjacent G residues, as others had reported in vivo. This was true independent of the activator, i.e., with MBP-NIFA and MBP-delta N-NIFA and with the homologous activator NTRC. When open complexes were formed with GTP as the activating nucleotide, the upstream G residue was probably as a consequence of initiation of transcription.
Berger
D K
DK
Department of Plant Biology, University of California, Berkeley 94720.
Narberhaus
F
F
Lee
H S
HS
Kustu
S
S
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D004252:DNA Mutational Analysis; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000235:genetics*; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation / Q000235:genetics; D009591:Nitrogenase / Q000096:biosynthesis / Q000235:genetics*; D010088:Oxidoreductases; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; C061338:NifA protein, Bacteria; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C084296:nifL protein, Bacteria; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase