EXTRAGENIC SUPPRESSORS OF THE TATA-BINDING PROTEIN

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The long-term objectives of the study are to define in molecular detail the mechanisms by which proteins regulate gene expression through direct interaction with the TATA-binding protein (TBP). TBP plays a central role in all eukaryotic gene expression since it is required for transcription initiation by all three nuclear RNA polymerase enzymes. A number of human and viral proteins that bind TBP, including the p53, myc and E1A proteins, have been implicated in transcriptional control and tumorigenesis. Thus an understanding of the means by which proteins bind TBP and regulate gene expression will have broad implications on diverse biological processes including growth control and cancer. We propose to investigate the mechanism by which the Std1 protein regulates gene expression as a paradigm for other transcriptional regulators that bind TBP. The STD1 gene was isolated as an extragenic suppressor of a dominant negative mutation in TBP. Std1p directly binds TBP as determined in both genetic and biochemical assays. Std1p enhances the DNA binding activity of TBP in a promoter specific fashion. Work from our lab and others indicate that Std1p activates gene expression but is not a typical transcriptional activator. Std1p does not contain a functional activation domain. Its binding site is not upstream of the TATA element, but overlaps the TATA element. All of these data combined suggest that the Std1 protein may regulate transcription by a mechanism that is fundamentally different from other transcriptional activators. Five specific aims are proposed. l) Characterize the Std1p-mediated regulation of TBP. The effect of Std1p on the kinetics, temperature dependence and site specificity of TBP-DNA and an intermediate of preinitiation complex formation (DAB) will be determined. The sequence specificity of Std1p's DNA binding activity and the role it plays in the regulation of the DNA binding activity of TBP will be determined. 2) The effect of Std1p on transcription initiation will be determined by nuclease S1 analysis of mRNA's from the SUC2 gene in vivo and from transcription reactions in vitro will be used to determine whether the Std1p alters the start site selection or increases the frequency of transcription initiation at the SUC2 promoter. 3) Identify and characterize the domains of Std1p that specify its known biochemical activities. These studies will use site specific mutagenesis of the protein and analysis of its biochemical properties in vitro and relate these activities with its function in vivo. 4) Isolate extragenic suppressors of std1 null alleles as a means to identify other protein that function in the same biochemical path way in vivo. 5) Isolate and characterize additional TBP-binding proteins as extragenic suppressors of the dominant negative allele of TBP used to isolate STD1. Also, temperature sensitive alleles of TBP with lesions in helix H2' will be screened for extragenic suppressors.