THE NITROGEN ASSIMILATION CONTROL PROTEIN, NAG, IS A DNA-BINDING TRANSCRIPTION ACTIVATOR IN KLEBSIELLA-AEROGENES

A 32-kDa polypeptide corresponding to NAG, the product of the Klebsiella aerogenes nac gene, was overexpressed from a plasmid carrying a tac'-'nac operon fusion and purified to near homogeneity by taking advantage of its unusual solubility properties. NAC was able to shift the electrophoretic migration of DNA fragments carrying the NAG-sensitive promoters hutUp, putPp1, and ureDp. The interaction between NAC and hutUp was localized to a 26-bp region centered approximately 64 bp upstream of the hutUp transcription initiation site. Moreover, NAC protected this region from DNase I digestion. Mobility shift and DNase I protection studies utilizing the putP and ureD promoter regions identified NAG-binding regions of sizes and locations similar to those found in hutUp. Comparison of the DNA sequences which were protected from DNase I digestion by NAC Suggests a minimal NAG-binding consensus sequence: 5'-ATA-N-9-TAT-3'. In vitro transcription assays demonstrated that NAG was capable of activating the transcription of hutUp by sigma(70)-RNA polymerase holoenzyme when this promoter was presented as either a linear or supercoiled DNA molecule. Thus, NAC displays the in vitro DNA-binding and transcription activation properties which have been predicted for the product of the nac gene.