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Sequential XylS-CTD binding to the Pm promoter induces DNA bending prior to activation

Lookup NU author(s): Dr Patricia Dominguez Cuevas

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Abstract

XylS protein, a member of the AraC family of transcriptional regulators, comprises a C-terminal domain (CTD) involved in DNA binding and an N-terminal domain required for effector binding and protein dimerization. In the absence of benzoate effectors, the N-terminal domain behaves as an intramolecular repressor of the DNA binding domain. To date, the poor solubility properties of the full-length protein have restricted XylS analysis to genetic approaches in vivo. To characterize the molecular consequences of XylS binding to its operator, we used a recombinant XylS-CTD variant devoid of the N-terminal domain. The resulting protein was soluble and monomeric in solution and activated transcription from its cognate promoter in an effector-independent manner. XylS binding sites in the Pm promoter present an intrinsic curvature of 35° centered at position -42 within the proximal site. Gel retardation and DNase footprint analysis showed XylS-CTD binding to Pm occurred sequentially: first a XylS-CTD monomer binds to the proximal site overlapping the RNA polymerase binding sequence to form complex I. This first event increased Pm bending to 50° and was followed by the binding of the second monomer, which further increased the observed global curvature to 98°. This generated a concomitant shift in the bending center to a region centered at position -51 when the two sites were occupied (complex II). We propose a model in which DNA structure and binding sequences strongly influence XylS binding events previous to transcription activation. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Publication metadata

Author(s): Domínguez-Cuevas P, Ramos J, Marqués S

Publication type: Article

Publication status: Published

Journal: Journal of Bacteriology

Year: 2010

Volume: 192

Issue: 11

Pages: 2682-2690

Print publication date: 01/06/2010

ISSN (print): 0021-9193

ISSN (electronic): 1067-8832

Publisher: American Society for Microbiology

URL: http://dx.doi.org/10.1128/JB.00165-10

DOI: 10.1128/JB.00165-10

PubMed id: 20363935

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