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Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4.

Abstract

Promoter-specific transcriptional activators (activators) stimulate transcription through direct interactions with one or more components of the transcription machinery, termed the "target." The identification of direct in vivo targets of activators has been a major challenge. Previous studies have provided evidence that the Tra1 subunit of the yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) complex is the target of the yeast activator Gal4. However, several other general transcription factors, in particular the mediator complex, have also been implicated as Gal4 targets. Here we perform a large-scale genetic screen to derive and characterize tra1 alleles that are selectively defective for interaction with Gal4 in vivo [Gal4 interaction defective (GID) mutants]. In contrast to WT Tra1, Tra1 GID mutants are not recruited by Gal4 to the promoter and cannot support Gal4-directed transcription, demonstrating the essentiality of the Gal4-Tra1 interaction. In yeast strains expressing a Tra1 GID mutant, binding of Gal4 to the promoter is unexpectedly also diminished, indicating that Gal4 and Tra1 bind cooperatively. Consistent with cooperative binding, we demonstrate that the Gal4-Tra1 interaction occurs predominantly on the promoter and not off DNA. Finally, we show that although Tra1 is targeted by other activators, these interactions are unaffected by GID mutations, revealing an unanticipated specificity of the Gal4-Tra1 interaction.

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  • Authors

    Lin L, Chamberlain L, Zhu LJ, Green MR

    Institution

    Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.

    Source

    Proceedings of the National Academy of Sciences of the United States of America 109:6 2012 Feb 7 pg 1997-2002

    MeSH

    Binding Sites
    Biological Assay
    DNA, Fungal
    DNA-Binding Proteins
    Fluorescence
    Galactokinase
    Galactose
    Gene Expression Regulation, Fungal
    Histone Acetyltransferases
    Mutation
    Promoter Regions, Genetic
    Protein Binding
    Saccharomyces cerevisiae
    Saccharomyces cerevisiae Proteins
    Trans-Activators
    Transcription Factors
    Transcriptional Activation

    Pub Type(s)

    Journal Article
    Research Support, N.I.H., Extramural
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    22308403