Mediator subunit Med15 dictates the conserved "fuzzy" binding mechanism of yeast transcription activators Gal4 and Gcn4.

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a "fuzzy" protein interface.

Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA.

Deletions within genes coding for subunits of the transcription coactivator SAGA caused strong genome-wide defects in transcription and SAGA-mediated chromatin modifications. In contrast, rapid SAGA depletion produced only modest transcription defects at 13% of protein-coding genes - genes that are generally more sensitive to rapid TFIID depletion. However, transcription of these 'coactivator-redundant' genes is strongly affected by rapid depletion of both factors, showing the overlapping functions of TFIID and SAGA at this gene set.

Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex.

Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes.

Transcription Activation Domains of the Yeast Factors Met4 and Ino2: Tandem Activation Domains with Properties Similar to the Yeast Gcn4 Activator.

Eukaryotic transcription activation domains (ADs) are intrinsically disordered polypeptides that typically interact with coactivator complexes, leading to stimulation of transcription initiation, elongation, and chromatin modifications. Here we examined the properties of two strong and conserved yeast ADs: Met4 and Ino2. Both factors have tandem ADs that were identified by conserved sequence and functional studies.

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface.

Although many transcription activators contact the same set of coactivator complexes, the mechanism and specificity of these interactions have been unclear. For example, do intrinsically disordered transcription activation domains (ADs) use sequence-specific motifs, or do ADs of seemingly different sequence have common properties that encode activation function? We find that the central activation domain (cAD) of the yeast activator Gcn4 functions through a short, conserved sequence-specific motif. Optimizing the residues surrounding this short motif by inserting additional hydrophobic residues creates very powerful ADs that bind the Mediator subunit Gal11/Med15 with high affinity via a "fuzzy" protein interface.

The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex.

The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation, allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft.

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

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.