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. 1995 Jun;177(12):3546–3555. doi: 10.1128/jb.177.12.3546-3555.1995

The nitrogen assimilation control protein, NAC, is a DNA binding transcription activator in Klebsiella aerogenes.

T J Goss 1, R A Bender 1
PMCID: PMC177061  PMID: 7768865

Abstract

A 32-kDa polypeptide corresponding to NAC, 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 NAC-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 NAC-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 NAC-binding consensus sequence: 5'-ATA-N9-TAT-3'. In vitro transcription assays demonstrated that NAC 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.

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Selected References

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