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

Abstract

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.

FIG. 1

Nucleotide changes introduced in the NAC-binding site from the hutU promoter and their effects on transcriptional activation. The clone designation is on the left. The wild-type sequence is shown at the top, and at the right are the β-galactosidase activities expressed in Miller units for each mutant grown under nitrogen excess (+N) or nitrogen limitation (−N) conditions. Values are the averages and standard deviations of at least three independent experiments. Ratio, −N/+N. Vertical lines indicate unchanged nucleotides. (A) Changes outside the 15-bp core. (B) Changes inside the 15-bp core.

FIG. 2

(A) Binding of NAC-binding site mutants from hutUp analyzed by gel mobility shift assay. The mutant NAC-binding sites were labeled with ³²P and incubated with NAC protein (0.28 μM). Bound and unbound fragments were separated by polyacrylamide gel electrophoresis. The radioactivity in each band was measured as described in Materials and Methods. (B) Correlation between protein binding and transcriptional activation for hutUp NAC-binding site mutants. The graph plots the activated levels of β-galactosidase (grown under nitrogen limitation) from Fig. 1B, and the binding percentages (normalized to clone V16 which was 49%) for the analyzed NAC binding-site mutants from panel A.

FIG. 3

Construction and transcriptional activation from two chimeric promoters. (A) Two different NAC-binding sites were placed upstream of the lacZ promoter (centered at −64). The 15-bp core of each NAC-binding site is indicated in bold type. The EcoRI restriction sites introduced by the mutagenic primers are underlined. (B) Transcriptional activation from the chimeric hutUp-lacZp and gdhAp-lacZp promoters. The bars indicate the specific activities of β-galactosidase for each construct under either nitrogen excess (+N) or nitrogen limitation (−N) conditions. Values are the averages of at least three independent experiments.

FIG. 4

NAC binding to the hutUp-lacZp and gdhAp-lacZp chimeric promoters. Restriction fragments from plasmids containing the NAC-binding sites were purified, labeled with ³²P, and incubated with NAC protein. Bound and unbound fragments were separated by polyacrylamide gel electrophoresis. The concentrations of purified NAC used were 0, 0.02, 0.04, 0.08, 0.17, 0.34, and 0.67 μM.

FIG. 5

DNase I footprints of NAC on the hutUp-lacZp and gdhAp-lacZp chimeric promoters. The plasmids containing the NAC-binding sites were purified, labeled, incubated with NAC protein, incubated with DNase I, and resolved in a polyacrylamide gel as explained in Materials and Methods. The final NAC concentrations used were 0, 0.134, and 0.670 μM. The first and last lanes show the products of Maxam-Gilbert sequencing reactions for the NAC targets on the hutUp-lacZp and gdhAp-lacZp promoter, respectively. Hypersensitive sites are indicated by arrows. The region protected by NAC in each construct is underlined.

FIG. 6

Transcriptional activation and protein binding in gdhA-lacZ chimeric promoters by NAC. Protein binding to the gdhAp-lacZp chimeric promoters is shown. Gel mobility shift assays were performed as described for Fig. 4 with purified NAC at a concentration of 0.28 μM.

FIG. 7

Transcriptional activation and protein binding in hutU-lacZ chimeric promoters. Protein binding to the hutUp-lacZp chimeric promoters is shown. Gel mobility shift assays were performed as described for Fig. 4 with purified NAC at a concentration of 0.34 μM.

FIG. 8

Functional nonequivalence of the half-sites in the NAC-binding site from hutUp. Each mutant site is drawn as composed by two half-sites. In the wild-type site (pCB648), the promoter-distal half-site is shaded and the promoter-proximal half-site is not. Each mutant site is a different combination of these two half-sites. The binding and activation values were taken from Table 2.