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 RECOGNITIONHELIX OF THE HTH MOTIF IN THE C-TERMINAL DOMAIN

Functional chimeras have been generated from the transcriptional activ ators VnfA and AnfA, which control expression of the alternative nitro genases in Azotobacter vinelandii. The activation profiles of the nati ve 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 t he promoter specificity of VnfA, confirming that the C-terminal domain contains the determinants of promoter specificity. However, substitut ing the VnfA sequence from the turn in the helix-turn-helix motif to t he C-terminus does not alter the promoter specificity of AnfA. These c hanges in promoter specificity were reflected in changes in affinity f or a VnfA-binding site, as measured by an in vivo repression assay usi ng a lacZ fusion to a synthetic promoter. This supports the assumption that promoter recognition is determined by activator binding to enhan cer-like sequences, and shows that the principal determinants of speci fic DNA-binding lie outside the 'recognition' helix. This may be a gen eral feature of transcriptional activators dependent on sigma(N)(sigma 54). The chimera with the promoter specificity of VnfA retained the de pendence on nitrogenase Fe protein characteristic of AnfA, indicating that this property is not related to particular promoter sequences, bu t is a function of the central or N-terminal domains of AnfA.