TOL plasmid transcription factor XylS binds specifically to the Pm operator sequence

Abstract

XylS, an AraC family transcription factor, positively regulates transcription of Pseudomonas putida TOL plasmid meta operon from the Pm promoter. A tandem of 15 bp homologous direct repeats, separated by 6 bp and overlapping with the -35 hexamer of the promoter, is required for the activation of Pm by XylS in vivo. In this study we have characterized specific binding of XylS to the Pm operator Om. XylS was overexpressed with an epitope tag in its N-terminus. Tagged XylS (N-XylS) was immunopurified and was shown to specifically bind to Om. We have used matrix-bound N-XylS in DNA footprinting and methylation interference experiments. Binding of N-XylS protects 44 bp in the Om region on both strands from DNase I digestion and generates hypersensitive sites (within the protected area) which lie on the same face of the DNA helix. Results of hydroxyl radical footprinting and methylation interference assays indicate that XylS binds along one side of the DNA and covers four helical turns. The protein has base-specific contacts in four adjacent major groove regions on the same helical face. Our data are in accord with the prediction of the presence of two separate DNA-binding units in an XylS molecule which are involved in base-specific contacts in two adjacent major-groove regions of a half-site. The direct repeat arrangement of the binding site and the mode of DNA binding of XylS are similar to the arrangement of recognition sites and the DNA contact pattern of AraC protein from Escherichia coli.

Critical Nucleotides in the Upstream Region of the XylS-dependent TOL meta-Cleavage Pathway Operon Promoter as Deduced from Analysis of Mutants

“…1). In favor of this proposal is the observation that a tagged XylS-protein immunoadsorbed onto glass beads produced footprints in vitro, which showed protection of the Gs within the above direct repeat at Ϫ48, Ϫ59, Ϫ60, and Ϫ69 (22). This is in agreement with our in vivo results.…”

RNA Polymerase Holoenzymes Can Share a Single Transcription Start Site for the Pm Promoter

“…Single point mutations in the binding site revealed that nucleotides located at Ϫ48 to Ϫ45 and at Ϫ58, Ϫ59, Ϫ61, and Ϫ69 are the most critical bases for appropriate XylS-Pm interactions (7)(8)(9). In vitro footprints obtained with a tagged XylS protein immunoadsorbed onto glass beads supported this organization (10). The arrangement of the two motifs is such that the proximal XylS binding site overlaps by 1 bp the RNA polymerase binding site at Ϫ35 (11) (Fig.…”

Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds

“…DNase I footprinting of the XylS interaction on the P m promoter showed a protected region from nucleotides Ϫ78 to Ϫ28 upstream of the transcription start site (102,103). The protein bound along one side of the DNA, covering four helices and thereby making specific contacts in four adjacent major grooves (102). By earlier sitedirected mutagenesis studies, a consensus direct repeat (TGCA-N 6 -GGNTA), which is present twice in the P m promoter between Ϫ35 and Ϫ49 and between Ϫ56 and Ϫ70, had been identified as the binding determinant (70,78,107).…”

“…This can be concluded from crystallographic data for the two AraC members Rob and MarA (114, 210). DNase I footprinting of the XylS interaction on the P m promoter showed a protected region from nucleotides Ϫ78 to Ϫ28 upstream of the transcription start site (102,103). The protein bound along one side of the DNA, covering four helices and thereby making specific contacts in four adjacent major grooves (102).…”

“…By earlier sitedirected mutagenesis studies, a consensus direct repeat (TGCA-N 6 -GGNTA), which is present twice in the P m promoter between Ϫ35 and Ϫ49 and between Ϫ56 and Ϫ70, had been identified as the binding determinant (70,78,107). In the presence of m-toluate, the degree of protection by XylS on the P m promoter increased (102,103), suggesting that the affinity of XylS to its binding site had changed. Subsequent in vivo methylation studies showed an altered methylation protection pattern of the XylS-bound region in the presence of m-toluate, which indicates that conformational changes take place under inducing conditions (78).…”