The nitrogen assimilation control protein, NAC, is a DNA binding transcription activator in Klebsiella aerogenes.
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.
Goss
T J
TJ
Department of Biology, University of Michigan, Ann-Arbor 48109-1048, USA.
Bender
R A
RA
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000502:physiology*; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D007711:Klebsiella pneumoniae / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000502:physiology; D014157:Transcription Factors / Q000302:isolation & purification / Q000502:physiology*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C072712:NAC protein, bacteria; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I