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J Biol Chem, Vol. 275, Issue 12, 9011-9018, March 24, 2000

Characterization of the N-terminal Domain of the Yeast Transcriptional Repressor Tup1
PROPOSAL FOR AN ASSOCIATION MODEL OF THE REPRESSOR COMPLEX Tup1·Ssn6^*

Carole Jabet^§, Elizabeth R. Sprague, Andrew P. VanDemark, and Cynthia Wolberger^§¶

From the  Department of Biophysics and Biophysical Chemistry and the ^§ Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

The yeast Tup1 and Ssn6 proteins form a transcriptional repression complex that represses transcription of a broad array of genes. It has been shown that the N-terminal domain of the Tup1 protein interacts with a region of the Ssn6 protein that consists of 10 tandem copies of a tetratricopeptide motif. In this work, we use a surface plasmon resonance assay to measure the affinity of the N-terminal domain of Tup1 for a minimal 3-TPR domain of Saccharomyces cerevisiae Ssn6 that is sufficient for binding to Tup1. This domain of Ssn6 binds with comparable affinity to S. cerevisiae and Candida albicans Tup1, but with 100-fold lower affinity to Tup1 protein containing a point mutation that gives rise to a defect in repression in vivo. Results from studies using analytical ultracentrifugation, CD spectroscopy, limited proteolysis, and ¹H NMR show that this domain of Tup1 is primarily -helical and forms a stable tetramer that is highly nonglobular in shape. X-ray diffraction recorded from poorly ordered crystals of the Tup1 tetramerization domain contains fiber diffraction typical of a coiled coil. Our results are used to propose a model for the structure of the N-terminal domain of Tup1 and its interaction with the Ssn6 protein.

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^* This work was supported by the National Science Foundation Grant MCB 98-08412 and by the Howard Hughes Medical Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 410-955-0728; Fax: 410-955-0637; E-mail: cwolberg@jhmi.edu.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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