Mechanism of Mediator recruitment by tandem Gcn4 activation domains and three Gal11 activator-binding domains.

Publication Type:

Journal Article

Source:

Molecular and cellular biology, Volume 30, Issue 10, p.2376-90 (2010)

Keywords:

2010, Basic Sciences Division, Basic-Leucine Zipper Transcription Factors, Center-Authored Paper, Gene Expression Regulation, Fungal, Genomics Core Facility, Mediator Complex, Protein Binding, Protein Structure, Tertiary, Protein Subunits, Proteomics Core Facility, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Shared Resources, TATA-Binding Protein Associated Factors, Transcription Factor TFIID, TRANSCRIPTIONAL ACTIVATION

Abstract:

Targets of the tandem Gcn4 acidic activation domains in transcription preinitiation complexes were identified by site-specific cross-linking. The individual Gcn4 activation domains cross-link to three common targets, Gal11/Med15, Taf12, and Tra1, which are subunits of four conserved coactivator complexes, Mediator, SAGA, TFIID, and NuA4. The Gcn4 N-terminal activation domain also cross-links to the Mediator subunit Sin4/Med16. The contribution of the two Gcn4 activation domains to transcription was gene specific and varied from synergistic to less than additive. Gcn4-dependent genes had a requirement for Gal11 ranging from 10-fold dependence to complete Gal11 independence, while the Gcn4-Taf12 interaction did not significantly contribute to the expression of any gene studied. Complementary methods identified three conserved Gal11 activator-binding domains that bind each Gcn4 activation domain with micromolar affinity. These Gal11 activator-binding domains contribute additively to transcription activation and Mediator recruitment at Gcn4- and Gal11-dependent genes. Although we found that the conserved Gal11 KIX domain contributes to Gal11 function, we found no evidence of specific Gcn4-KIX interaction and conclude that the Gal11 KIX domain does not function by specific interaction with Gcn4. Our combined results show gene-specific coactivator requirements, a surprising redundancy in activator-target interactions, and an activator-coactivator interaction mediated by multiple low-affinity protein-protein interactions.