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Originally published In Press as doi:10.1074/jbc.M010665200 on January 19, 2001

J. Biol. Chem., Vol. 276, Issue 15, 11883-11894, April 13, 2001
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High Affinity Interaction of Yeast Transcriptional Regulator, Mot1, with TATA Box-binding Protein (TBP)*

Joanne I. AdamkewiczDagger , Karin E. HansenDagger , Wendy A. Prud'homme§, Jennifer L. Davis, and Jeremy Thorner||

From the Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202

Yeast Mot1, an essential ATP-dependent regulator of basal transcription, removes TATA box-binding protein (TBP) from TATA sites in vitro. Complexes of Mot1 and Spt15 (yeast TBP), radiolabeled in vitro, were immunoprecipitated with anti-TBP (or anti-Mot1) antibodies in the absence of DNA, showing Mot1 binds TBP in solution. Mot1 N-terminal deletions (residues 25-801) abolished TBP binding, whereas C-terminal ATPase domain deletions (residues 802-1867) did not. Complex formation was prevented above 200 mM salt, consistent with electrostatic interaction. Correspondingly, TBP variants lacking solvent-exposed positive charge did not bind Mot1, whereas a mutant lacking positive charge within the DNA-binding groove bound Mot1. ATPase-defective mutant, Mot1(D1408N), which inhibits growth when overexpressed (but is suppressed by co-overexpression of TBP), bound TBP normally in vitro, suggesting it forms nonrecyclable complexes. N-terminal deletions of Mot1(D1408N) were not growth-inhibitory. C-terminal deletions were toxic when overexpressed, and toxicity was ameliorated by TBP co-overproduction. Residues 1-800 of Mot1 are therefore necessary and sufficient for TBP binding. The N terminus of 89B, a tissue-specific Drosophila Mot1 homolog, bound the TBP-like factor, dTRF1. Native Mot1 and derivatives deleterious to growth localized in the nucleus, whereas nontoxic derivatives localized to the cytosol, suggesting TBP binding and nuclear transport of Mot1 are coupled.

* This work was supported by a predoctoral fellowship from the Howard Hughes Medical Institute (to J. I. A.), by a University fellowship from the Graduate Division of the University of California, Berkeley, and a predoctoral fellowship from the National Science Foundation (to K. E. H.), by a University of California President's Undergraduate research fellowship (to W. A. P.), by Postdoctoral Fellowship PF-3308 from the American Cancer Society, California Division (to J. L. D.), and by Research Grant GM21841 from the National Institutes of Health and facilities provided by the Berkeley campus Cancer Research Laboratory (to J. T.).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.

Dagger Both authors contributed equally to this work.

§ Current address: Dept. of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139.

Current address: Cell Genesys, Inc., 342 Lakeside Dr., Foster City, CA 94404.

|| To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, Rm. 401, Barker Hall, University of California, Berkeley, CA 94720-3202. Tel.: 510-642-2558; Fax: 510-643-6791; E-mail: jeremy@socrates.berkeley.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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