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Mol Cell Biol, July 1998, p. 3752-3761, Vol. 18, No. 7
Department of Biological Chemistry,
University of California
Received 20 October 1997/Returned for modification 21 November
1997/Accepted 29 March 1998
Previous in vitro studies have shown that initiation of
transcription of ribosomal DNA (rDNA) in the yeast Saccharomyces
cerevisiae involves an interaction of upstream activation factor
(UAF) with the upstream element of the promoter, forming a stable
UAF-template complex; together with TATA-binding protein (TBP), UAF
then recruits an essential factor, core factor (CF), to the promoter,
forming a stable preinitiation complex. TBP interacts with both UAF and CF in vitro. In addition, a subunit of UAF, Rrn9p, interacts with TBP
in vitro and in the two-hybrid system, suggesting the possible importance of this interaction for UAF function. Using the yeast two-hybrid system, we have identified three mutations in
RRN9 that abolish the interaction of Rrn9p with TBP without
affecting its interaction with Rrn10p, another subunit of UAF. Yeast
cells containing any one of these individual mutations,
L110S, L269P, or L274Q, did not
show any growth defects. However, cells containing a combination of
L110S with one of the other two mutations showed a
temperature-sensitive phenotype, and this phenotype was suppressed by
fusing the mutant genes to SPT15, which encodes TBP. In
addition, another mutation (F186S), which disrupts both
Rrn9p-TBP and Rrn9p-Rrn10p interactions in the two-hybrid system,
abolished UAF function in vivo, and this mutational defect was
suppressed by fusion of the mutant gene to SPT15 combined
with overexpression of Rrn10p. These experiments demonstrate that the
interaction of UAF with TBP, which is presumably achieved by the
interaction of Rrn9p with TBP, is indeed important for high-level
transcription of rDNA by RNA polymerase I in vivo.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Interaction of TATA-Binding Protein with Upstream Activation
Factor Is Required for Activated Transcription of Ribosomal DNA by
RNA Polymerase I in Saccharomyces cerevisiae In
Vivo
Irvine, Irvine, California 92697-1700
*
Corresponding author. Mailing address: Department of
Biological Chemistry, University of California
Irvine, Irvine, CA
92697-1700. Phone: (949) 824-4565. Fax: (949) 824-3201. E-mail:
mnomura{at}uci.edu.
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