Nitrogen Assimilation Control Protein Research Articles

Two roles for the DNA recognition site of the Klebsiella aerogenes nitrogen assimilation control protein.

The nitrogen assimilation control protein (NAC) binds to a site within the promoter region of the histidine utilization operon (hutUH) of Klebsiella aerogenes, and NAC bound at this site activates transcription of hutUH. This NAC-binding site was characterized by a combination of random and directed DNA mutagenesis. Mutations that abolished or diminished in vivo transcriptional activation by NAC were found to lie within a 15-bp region contained within the 26-bp region protected by NAC from DNase I digestion. This 15-bp core has the palindromic ends ATA and TAT, and it matches the consensus for LysR family transcriptional regulators. Protein-binding experiments showed that transcriptional activation in vivo decreased with decreasing binding in vitro. In contrast to the NAC-binding site from hutUH, the NAC-binding site from the gdhA promoter failed to activate transcription from a semisynthetic promoter, and this failure was not due to weak binding or greatly distorted protein-DNA structure. Mutations in the promoter-proximal half-site of the NAC-binding site from gdhA allowed this site to activate transcription. Similar studies using the NAC-binding site from hut showed that two mutations in the promoter proximal half-site increased binding but abolished transcriptional activation. Interestingly, for symmetric mutations in the promoter-distal half-site, loss of transcriptional activation was always correlated with a decrease in binding. We conclude from these observations that if the binding in vitro reflects the binding in vivo, then binding of NAC to DNA is not sufficient for transcriptional activation and that the NAC-binding site can be functionally divided in two half-sites, with related but different functions.

Genetic analysis, using P22 challenge phage, of the nitrogen activator protein DNA-binding site in the Klebsiella aerogenes put operon.

The nac gene product is a LysR regulatory protein required for nitrogen regulation of several operons from Klebsiella aerogenes and Escherichia coli. We used P22 challenge phage carrying the put control region from K. aerogenes to identify the nucleotide residues important for nitrogen assimilation control protein (NAC) binding in vivo. Mutations in an asymmetric 30-bp region prevented DNA binding by NAC. Gel retardation experiments confirmed that NAC specifically binds to this sequence in vitro, but NAC does not bind to the corresponding region from the put operon of Salmonella typhimurium, which is not regulated by NAC.

Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor

A chimeric promoter with the nitrogen assimilation control protein binding site from hutUp of Klebsiella aerogenes fused to the lacZ core promoter from Escherichia coli was built and cloned in a lacZ reporter plasmid. This construct showed a 14-fold increase of beta-galactosidase activity upon nitrogen limitation. Primer extension experiments showed that the nitrogen assimilation control protein activates lacZp1 in a position-dependent manner.

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.