Construction of chimeric proteins from the sigma N-associated transcriptional activators VnfA and AnfA of Azotobacter vinelandii shows that the determinants of promoter specificity lie outside the 'recognition' helix of the HTH motif in the C-terminal domain.
Functional chimeras have been generated from the transcriptional activators VnfA and AnfA, which control expression of the alternative nitrogenases in Azotobacter vinelandii. The activation profiles of the native and chimeric proteins have been determined using lacZ fusions to A. vinelandii anf and vnf promoters in Klebsiella pneumoniae. Replacing the C-terminal domain of AnfA with that of VnfA gives a protein with the promoter specificity of VnfA, confirming that the C-terminal domain contains the determinants of promoter specificity. However, substituting the VnfA sequence from the turn in the helix-turn-helix motif to the C-terminus does not alter the promoter specificity of AnfA. These changes in promoter specificity were reflected in changes in affinity for a VnfA-binding site, as measured by an in vivo repression assay using a lacZ fusion to a synthetic promoter. This supports the assumption that promoter recognition is determined by activator binding to enhancer--like sequences, and shows that the principal determinants of specific DNA-binding lie outside the 'recognition' helix. This may be a general feature of transcriptional activators dependent on sigma N (sigma 54). The chimera with the promoter specificity of VnfA retained the dependence on nitrogenase Fe protein characteristic of AnfA, indicating that this property is not related to particular promoter sequences, but is a function of the central or N-terminal domains of AnfA.
Jacob
J
J
AFRC IPSR Nitrogen Fixation Laboratory, University of Sussex, Brighton, UK.
Drummond
M
M
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005798:Genes, Bacterial; D007763:Lac Operon; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation / Q000235:genetics; D009591:Nitrogenase / Q000235:genetics; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D015534:Trans-Activators; D009591:Nitrogenase; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54