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J. Biol. Chem., Vol. 275, Issue 28, 21158-21168, July 14, 2000
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From the Department of Molecular and Cell Biology, Division of
Biochemistry and Molecular Biology, University of California,
Berkeley, California 94720-3202
The 1867-residue Mot1 protein is a
member of a superfamily of ATPases, some of which are helicases, that
interact with protein-nucleic acid assemblies. Mot1 is an essential
regulator of RNA polymerase II-dependent transcription
in vivo and dissociates TATA box-binding protein (TBP)-DNA
complexes in vitro. Mot1-(His)6 was purified to
apparent homogeneity from yeast extracts. The preparation efficiently dissociated TBP·TATA complexes, suggesting that no other protein or
cofactor is required. Mot1 behaved as a non-globular monomer in
hydrodynamic studies, and no association was detected between differentially tagged co-expressed Mot1 constructs. ATPase activity was
stimulated about 10-fold by high ionic strength or alkaline pH, or by
deletion of the N-terminal TBP-binding segment, suggesting that the
N-terminal domain negatively regulates the C-terminal ATPase domain
(Mot1C). Correspondingly, at moderate salt concentration, Mot1 ATPase
(but not Mot1C) was stimulated
Purification and Enzymic Properties of Mot1 ATPase, a Regulator
of Basal Transcription in the Yeast Saccharomyces
cerevisiae*
,
10-fold by yeast TBP, suggesting that
interaction with TBP relieves a conformational constraint in Mot1.
Double- or single-stranded TATA-containing DNA did not affect ATPase
activity of Mot1 or Mot1C, with or without TBP. Mot1 did not exhibit
detectable helicase activity in strand displacement assays using
substrates with flush ends or 5'- or 3'-overhangs. Mot1-catalyzed
dissociation of TBP from DNA was not prevented by a psoralen cross-link
positioned immediately preceding the TATA sequence. Thus, Mot1 most
likely promotes release of TBP from TATA-containing DNA by causing a
structural change in TBP itself, rather than by strand unwinding.
*
This work was supported by a predoctoral fellowship from the
Howard Hughes Medical Institute (to J. I. A.), by Postdoctoral Fellowship LT-316/92 from the Human Frontier Science Program
Organization (to C. G. F. M.), by a university fellowship from the
graduate division of the University of California, Berkeley, by 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 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.
Current address: Institut Curie, INSERM U255, 26 Rue d'Ulm, Paris
75005, France.
§
Current address: Dept. of Chemical Engineering, Massachusetts
Institute of Technology, Cambridge, MA 02139.
¶
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; and E-mail:
jeremy@socrates.berkeley.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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