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Molecular and Cellular Biology, April 1999, p. 3039-3050, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The SKN-1 Amino-Terminal Arm Is a DNA Specificity Segment

Thiphaphone Kophengnavong, Adam S. Carroll, and T. Keith Blackwell*

Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

Received 6 August 1998/Returned for modification 21 September 1998/Accepted 14 January 1999

The Caenorhabditis elegans SKN-1 protein binds DNA through a basic region like those of bZIP proteins and through a flexible amino-terminal arm segment similar to those with which numerous helix-turn-helix proteins bind to bases in the minor groove. A recent X-ray crystallographic structure suggests that the SKN-1 amino-terminal arm provides only nonspecific DNA binding. In this study, however, we demonstrate that this segment mediates recognition of an AT-rich element that is part of the preferred SKN-1 binding site and thereby significantly increases the sequence specificity with which SKN-1 binds DNA. Mutagenesis experiments show that multiple amino acid residues within the arm are involved in binding. These residues provide binding affinity through distinct but partially redundant interactions and enhance specificity by discriminating against alternate sites. The AT-rich element minor groove is important for binding of the arm, which appears to affect DNA conformation in this region. This conformational effect does not seem to involve DNA bending, however, because the arm does not appear to affect a modest DNA bend that is induced by SKN-1. The data illustrate an example of how a small, flexible protein segment can make an important contribution to DNA binding specificity through multiple interactions and mechanisms.

* Corresponding author. Mailing address: Center for Blood Research, Harvard Medical School, Boston, MA 02115. Phone: (617) 278-3150. Fax: (617) 278-3131. E-mail: blackwell{at}cbr.med.harvard.edu.

Molecular and Cellular Biology, April 1999, p. 3039-3050, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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