The long-term objectives of this application are to learn more about fundamental aspects of transcription initiation in eukaryotes. This process is strongly conserved in all eukaryotes, ranging from yeast to human. The studies will focus on protein factors discovered in the yeast, Saccharomyces cerevisiae, that control the activity of TATA-binding protein (TBP). TBP is a highly conserved protein, found throughout eukaryotes, that is essential for transcription initiation by all three nuclear RNA polymerases. The studies in this application are focused on transcription initiation by RNA polymerase II. A combination of genetic and biochemical studies have shown that two yeast transcription factors, Mot1p and Spt3p, act as negative and positive regulators, respectively, of TBP activity. Mot1p is an essential function that can remove TBP from its normal DNA binding site, a TATA box. Spt3p is a nonessential function; its activity may be to counteract Mot1p activity at particular promoters, thereby conferring promoter-specificity on TBP binding to DNA. The proposed experiments are to use genetic, molecular, and biochemical approaches to analyze Spt3p and its relationship with Mot1p, and to study how these and other related transcription factors interact with TBP to control transcription initiation. First, the interaction between Spt3p and Mot1p will be examined by several approaches, including the isolation of new mot1 mutations that affect the Mot1p-Spt3p interaction, a direct test for Mot1p-Spt3p physical interaction, and in vitro transcription experiments to attempt to define the roles of these functions and their interactions in transcription. Second, other important aspects of Spt3p will be elucidated by in vivo footprinting of an Spt3p-dependent promoter, mutational analysis of an Spt3p-dependent promoter, and the isolation and analysis of Spt3p dominant-negative mutations. Finally, other functions related to Spt3p will be studied, including three factors identified in a recent mutant hunt for new SPT genes, and factors to be identified by a screen for other Spt3p-like factors that interact with Mot1p. These studies should reveal important aspects of transcriptional control in yeast. Given the conservation between yeast and humans, the results from these studies will be applicable to transcription in humans. These studies are relevant to human disease, since altered transcription in humans has been implicated in diseases such as cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045720-07
Application #
2415150
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1991-05-01
Project End
1999-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
7
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Harvard University
Department
Genetics
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
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Neumüller, Ralph A; Gross, Thomas; Samsonova, Anastasia A et al. (2013) Conserved regulators of nucleolar size revealed by global phenotypic analyses. Sci Signal 6:ra70
Ahn, Sejin; Spatt, Dan; Winston, Fred (2012) The Schizosaccharomyces pombe inv1(+) regulatory region is unusually large and contains redundant cis-acting elements that function in a SAGA- and Swi/Snf-dependent fashion. Eukaryot Cell 11:1067-74
Rando, Oliver J; Winston, Fred (2012) Chromatin and transcription in yeast. Genetics 190:351-87
Helmlinger, Dominique; Marguerat, Samuel; Villen, Judit et al. (2011) Tra1 has specific regulatory roles, rather than global functions, within the SAGA co-activator complex. EMBO J 30:2843-52
Hickman, Mark J; Spatt, Dan; Winston, Fred (2011) The Hog1 mitogen-activated protein kinase mediates a hypoxic response in Saccharomyces cerevisiae. Genetics 188:325-38
Winston, Fred (2009) A transcription switch toggled by noncoding RNAs. Proc Natl Acad Sci U S A 106:18049-50
Helmlinger, Dominique; Marguerat, Samuel; Villen, Judit et al. (2008) The S. pombe SAGA complex controls the switch from proliferation to sexual differentiation through the opposing roles of its subunits Gcn5 and Spt8. Genes Dev 22:3184-95
Laprade, Lisa; Rose, David; Winston, Fred (2007) Characterization of new Spt3 and TATA-binding protein mutants of Saccharomyces cerevisiae: Spt3 TBP allele-specific interactions and bypass of Spt8. Genetics 177:2007-17
Hickman, Mark J; Winston, Fred (2007) Heme levels switch the function of Hap1 of Saccharomyces cerevisiae between transcriptional activator and transcriptional repressor. Mol Cell Biol 27:7414-24

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