Purification and in vitro activity of a truncated form of ANFA. Transcriptional activator protein of alternative nitrogenase from Azotobacter vinelandii.
The ANFA protein is the transcriptional activator of the sigma 54-dependent anfHDGK operon, which codes for the structural genes of the third nitrogenase system in Azotobacter vinelandii. We have purified, in soluble active form, an N-terminally truncated form of the protein, delta ANFA, which activates transcription from the anfH promoter and other sigma 54-dependent promoters in a purified transcription system. Sequences upstream of the anfH promoter and the presence of the integration host factor protein stimulate transcription, and we have shown that delta ANFA binds to sites situated between 200 and 300 base pairs upstream of the anfH promoter. In common with other sigma 54-dependent activators, ANFA has a highly conserved ATP binding motif in its central domain, and we have demonstrated that ATP or GTP is required for productive complex formation and that the purified truncated protein has a constitutive ATPase activity, which is presumably required to drive open complex formation.
Austin
S
S
Institute of Plant Science Research, Nitrogen Fixation Laboratory, University of Sussex, Brighton, United Kingdom.
Lambert
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005798:Genes, Bacterial; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009591:Nitrogenase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012995:Solubility; D015534:Trans-Activators / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D009591:Nitrogenase; C070293:nitrogenase 3; D000251:Adenosine Triphosphatases