IN-VITRO STUDIES OF THE DOMAINS OF THE NITROGEN-FIXATION REGULATORY PROTEIN NIFA

The prokaryotic enhancer-binding protein NIFA is a multidomain transcr iptional activator that catalyzes the formation of open complexes at n itrogen fixation (nif) promoters by a specialized form of RNA polymera se containing sigma 54. The NIFA protein from Klebsiella pneumoniae co nsists of three domains: the N-terminal domain of unknown function; th e central catalytic domain, which is sufficient for transcriptional ac tivation; and the C-terminal DNA-binding domain. Purified fusion prote ins between maltose-binding protein (MBP) and NIFA deleted of its N-te rminal domain (MBP-Delta N-NIFA) or its C-terminal domain (MBP-NIFA-De lta C) activated transcription from the K. pneumoniae nifH promoter bo th in vitro and in vivo, We previously showed that the same was true f or a fusion between MBP and the central domain of NIFA. These results indicate that NIFA is sufficiently modular for all fusions carrying it s catalytic domain to be active. Unexpectedly, however, simple predict ions regarding the location of determinants of the heat lability and i nsolubility of NIFA, which were based on previous studies of its isola ted 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 eith er of two adjacent G residues, as others had reported in vivo. This wa s true independent of the activator, i.e., with MBP-NIFA and MBP-Delta N-NIFA and with the homologous activator NTRC. When open complexes we re formed with GTP as the activating nucleotide, the upstream G residu e was favored and the complexes formed were distinguishable from those formed in the presence of other nucleotides, probably as a consequenc e of initiation of transcription,