IDENTIFICATION OF SEQUENCES IMPORTANT FOR RECOGNITION OF VNF GENES BYTHE VNFA TRANSCRIPTIONAL ACTIVATOR IN AZOTOBACTER-VINELANDII

To analyze regulation of the vanadium-dependent nitrogenase of Azotoba cter vinelandii, plasmids carrying vnfE-, vnfH-, or vnfD-lacZ fusions were transferred to Escherichia coli. These genes were expressed only if VnfA was present. Deletions of the vnfE upstream region were constr ucted and comparison of a region necessary for expression with sequenc es upstream of other unf genes indicated a substantially conserved mot if, GTAC-N6-GTAC, hypothesized to be the binding site for VnfA. This m otif was duplicated with 17 or 18 bases lying between each in the vnfH and unfD promoters. Deletion analysis of the vnfH promoter indicated that both motifs were necessary for full expression. In footprinting e xperiments, VnfA significantly protected from methylation the guanine residues within or immediately adjacent to the proposed VnfA recogniti on motifs. The active form of VnfA is probably interacting dimers, a t etramer, or a higher order oligomer since two regions of dyad symmetry are required for its interaction with the DNA.