Differential DNA binding of transcriptional regulator PcaU from Acinetobacter sp. strain ADP1

Abstract

Transcriptional regulator PcaU from Acinetobacter sp. strain ADP1 governs expression of genes for protocatechuate degradation (pca genes) as a repressor or an activator depending on the levels of the inducer protocatechuate and of its own gene. PcaU is a member of the IclR protein family. Here the DNA binding properties of the purified protein are described in terms of the location of the binding sites and the affinity to these sites. Native PcaU was purified after overexpression of the pcaU gene in Escherichia coli. It is a dimer in solution. The binding site in the pcaU-pcaI intergenic region is located between the two divergent promoters covering 45 bp, which includes three perfect 10-bp repetitions. A PcaU binding site downstream of pcaU is covered by PcaU across two palindromic sequence repetitions. The affinity of PcaU for the intergenic binding sites is 50-fold higher (dissociation constant [K(d)], 0.16 nM) than the affinity for the site downstream of pcaU (K(d), 8 nM). The binding of PcaU was tested after modifications of the intergenic binding site. Removal of any external sequence repetition still allowed for specific binding of PcaU, but the affinity was significantly reduced, suggesting an important role for all three sequence repetitions in gene expression. The involvement of DNA bending in the regulatory process is suggested by the observed strong intrinsic curvature displayed by the pcaU-pcaI intergenic DNA.

FIG. 1.

Intergenic region between pcaU and pcaI from Acinetobacter sp. strain ADP1. Dashed arrows, three 10-bp sequence repetitions located in the binding site of regulator protein PcaU; bracket, region protected from DNase I footprinting by PcaU; boxes, stretches of at least three successive adenosine residues. The distance from each stretch of As to the next one is given by a number underneath the sequence (printed open if the distance corresponds to one or multiple DNA helix turns). Vertical arrow, bending center for the 128-bp Bst UI-EcoRI fragment.

FIG. 2.

(A) Denaturing SDS-PAGE from fractions of the purification of native PcaU after overexpression in E. coli. Lane 2, cell extract of the host strain with pET-21(+) without insert; lane 3, cell extract of the strain overproducing PcaU; lane 4, fraction after anion exchange chromatography; lane 5, fraction after affinity chromatography on heparin resin; lane 1, size standard. A low-molecular-weight protein still present in the preparation after chromatography on heparin was separated from PcaU by gel filtration (not shown). (B) Size determination of the native PcaU protein by gel filtration on Superdex 75. Squares, K_av values for reference proteins of known size; vertical line, respective value for PcaU.

FIG. 3.

Comparison of the two PcaU binding sites characterized here and the binding site of the closest homologue of PcaU, PobR from Acinetobacter sp. strain ADP1. Genes are represented as boxes with arrowheads inside indicating the direction of transcription. Arrows above sequence, sequence repetitions. Sequence identity between the two PcaU binding sites is highlighted by shaded boxes; identity between the pca and pob intergenic binding sites is shown by small open boxes. Large open boxes indicate the areas protected by the respective regulators in DNase I footprinting experiments.

FIG. 4.

Determination of the PcaU binding sites by DNase I footprinting in the pcaU-pcaI intergenic region (A) and downstream of pcaU (B). PcaU was added in various amounts (20, 10, 5, and 2.5 ng)

FIG. 5.

Determination of the K_d for PcaU binding to the pcaU-pcaI intergenic region (A) and to the binding site downstream of pcaU (B). The K_d was the PcaU concentration at which 50% of the DNA was bound. For the downstream binding site, a maximum of 50% of the total probe was bound under any condition; thus the K_d was the PcaU concentration at which 25% of the DNA was bound.

FIG. 6.

Qualities of purified PcaU binding to modified intergenic binding sites. The relevant part of the respective DNA fragment used for the gel retardation assay is shown on the left. The wild-type motif was contained on a 214-bp EcoRI-XmnI fragment, the motif containing only the direct sequence repetitions was contained on a 45-bp fragment cloned into plasmid pBend5, and the motif containing only the palindrome was contained on a derivative of pZR18 after removal of the 218-bp SwaI-Eco47III fragment containing the direct sequence repetition. Results of the respective gel retardation assays are shown on the right. The amount of unshifted probe was plotted against the respective PcaU concentration and served for determination of K_d.

FIG. 7.

Determination of DNA bending within the 128-bp BstUI-EcoRI fragment from the pcaU-pcaI intergenic region (Fig. 1) by a circular permutation assay using bending vector pBend5. (Top) The relevant part of plasmid p5/128 is shown with the restriction sites of the permutation element and the test DNA as a box in the middle. Horizontal arrows, locations of the PcaU binding sites; vertical arrow, bending center revealed in this experiment. Numbers under the left half of the permutation element, distances in base pairs between the BstUI site of the test DNA and the left end of the fragment resulting from restriction digestion with the respective enzymes. These numbers were used as the distances from left end to insert in the graph (bottom). (Middle) Example of a gel containing samples of plasmid p5/128 after restriction cleavage with the enzymes indicated. The permutated fragments had a length of 255 bp; for comparison two fragments of the standard are indicated. (Bottom) Plot of the relative mobilities of the fragments against the distance between the left end of the permutated fragments and the test DNA enables the determination of the bending center. Relative mobilities are the averages of six gels; the error was between 0.7 and 1.5%.