Characterization of new Spt3 and TATA-binding protein mutants of Saccharomyces cerevisiae: Spt3 TBP allele-specific interactions and bypass of Spt8.

The Spt-Ada-Gcn5-acetyltransferase (SAGA) complex of Saccharomyces cerevisiae is a multifunctional coactivator complex that has been shown to regulate transcription by distinct mechanisms. Previous results have shown that the Spt3 and Spt8 components of SAGA regulate initiation of transcription of particular genes by controlling the level of TATA-binding protein (TBP/Spt15) associated with the TATA box. While biochemical evidence exists for direct Spt8-TBP interactions, similar evidence for Spt3-TBP interactions has been lacking. To learn more about Spt3-TBP interactions in vivo, we have isolated a new class of spt3 mutations that cause a dominant-negative phenotype when overexpressed. These mutations all cluster within a conserved region of Spt3. The isolation of extragenic suppressors of one of these spt3 mutations has identified two new spt15 mutations that show allele-specific interactions with spt3 mutations with respect to transcription and the recruitment of TBP to particular promoters. In addition, these new spt15 mutations partially bypass an spt8 null mutation. Finally, we have examined the level of SAGA-TBP physical interaction in these mutants. While most spt3, spt8, and spt15 mutations do not alter SAGA-TBP interactions, one spt3 mutation, spt3-401, causes a greatly increased level of SAGA-TBP physical association. These results, taken together, suggest that a direct Spt3-TBP interaction is required for normal TBP levels at Spt3-dependent promoters in vivo.

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