Chimeric transcriptional activators generated in vivo from VnfA and AnfA of Azotobacter vinelandii: N-terminal domain of AnfA is responsible for dependence on nitrogenase Fe protein.
In vivo recombinants generating chimeras between the transcriptional activators VnfA and AnfA of Azotobacter vinelandii were constructed by cloning their structural genes in tandem and selecting against a conditionally lethal gene inserted between them. The parent molecules differ in their promoter specificities and in that AnfA, but not VnfA, requires the Fe protein of nitrogenase for its activity. Chimeras with fusion junctions in the N-terminal half of the central domain were found to be inactive, probably as a result of misfolding. All chimeras carrying the C-terminal domain of AnfA showed the corresponding promoter specificity, supporting the model which ascribes promoter specificity to the DNA-binding properties of the C-terminal domain. None of the chimeras showed the dependence on Fe protein typical of AnfA, including one which composed 82% of AnfA with only a short segment of VnfA at the N terminus. Deleting the N-terminal domain of AnfA gave a fully active protein which was also independent of Fe protein. This indicates that the N-terminal domain has an inhibitory effect on activity which is relieved by Fe protein.
AFRC IPSR Nitrogen Fixation Laboratory, University of Sussex, Brighton, United Kingdom.
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D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009591:Nitrogenase / Q000378:metabolism*; D010088:Oxidoreductases; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C090095:VnfA protein, Azotobacter vinelandii; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase