Comparison of Tra1 and TRRAP domains. (A) Modular structure of the Tra1/TRRAP protein. A representative three-dimensional protein structure for each of the five domains is depicted below each arrow with the corresponding Protein Data Bank code. (B) Alignment of predicted domain and repeat architectures of Tra1 and TRRAP. Individual rectangles in the HEAT and FAT domains represent single HEAT and TPR repeats, respectively. Dark gray rectangles represent predicted disordered regions, and light gray rectangles denote a non-repeat-containing segment. Gaps in the alignment are indicated by a line. ID, identity.

Summary of TRA1 deletions and single-amino-acid substitutions in the PI3K domain and their roles in SAGA and NuA4 coactivator function. (A) Schematic of the repeat and domain architecture of Tra1. The color scheme and structural features are the same as in Fig. . The deletion name and location are indicated to the left of each bracket. Inviable Tra1 deletions (all causing both SAGA and NuA4 complex integrity defects) are indicated by red lettering, and viable deletions are indicated by blue lettering. Every fifth repeat is denoted by an asterisk or dot in the HEAT and FAT domains, respectively. (B) Surface representation of the PI3K domain of Tra1 based on the crystal structure of 3ihy. The N-terminal and C-terminal lobes of the Tra1 PI3K domain model are colored in white and gray, respectively. Inviable amino acid substitutions are colored red, and viable substitutions are blue.

Growth phenotypes of selected TRA1 deletion mutants. (Left panels) Viable TRA1 mutants from Table S1 in the supplemental material were compared with a wild-type strain for growth on glucose complete (GC) medium lacking tryptophan, isoleucine, and valine incubated at 30°C with or without 3 μg/ml SMM. (Right panels) The indicated Tra1 mutations were incubated on GC medium and grown at the indicated temperatures. Fivefold serial dilutions of cells were spotted onto plates.

Effect of TRA1 mutations on SAGA and NuA4 complex integrity. Western analysis of immunopurified Flag-Tra1. Flag-Tra1 complexes were purified from whole-cell extract and analyzed by Western blotting using the following antibodies: Flag, Ada1, Spt3, Gcn5, Taf12, Esa1, and Yaf9. Tra1 deletions that give rise to a truncated protein are indicted by an asterisk (Δ8 is significantly smaller than wild-type Tra1 and is not visible on this blot).

Effect of SAGA and NuA4 mutations on transcription. Strains containing either wild type (WT) or the indicated SAGA or NuA4 subunit deletions were grown under conditions that induce Rap1-dependent genes (A), Gcn4-dependent genes (B), or Gal4-dependent genes (C). Total cellular RNA was prepared and quantitated by RT-qPCR, and results are represented relative to the wild-type value set at 1.0. UI, not induced. Experiments were performed in biological duplicate, and error bars represent standard deviations.

Effect of TRA1 mutations on transcription. Strains containing either wild-type TRA1 or the indicted TRA1 deletions were grown under conditions that induce Rap1-dependent genes (A to C), Gcn4-dependent genes (D to F), or Gal4-dependent genes (G to I). Total cellular RNA was prepared from these cells and quantitated by RT-qPCR, and results are represented relative to the wild-type value set at 1.0. Brackets below the TRA1 mutant labels indicate the domain(s) disrupted by the deletion. P/Fc, junction between PI3K C-terminal lobe and FATC domains. Experiments were performed in biological triplicate, and errors bars represent standard deviations.

Effect of TRA1 mutations on recruitment of SAGA and NuA4 to promoters. Strains were grown as described for Fig. , cross-linked, and analyzed by ChIP: activator (A, E, and I), Rpb1 (B, F, and J), Flag-Tra1 (C, G, and K), Eaf1-TAP (D), and Spt7-TAP (H and L). ChIP results are represented relative to the wild-type value, set at 1.0. Different promoters analyzed by RT-qPCR are indicated. UI, not induced; NT, no epitope tag. Brackets below the TRA1 mutant labels indicate the domain(s) disrupted by the deletion. P/Fc, junction between PI3K C-terminal lobe and FATc domains. Experiments were performed in biological triplicate, and error bars represent standard deviations.

TRA1 mutations affect the binding of SAGA and NuA4 complexes to GST-Gcn4 fusion protein. Equivalent amounts of purified Flag-Tra1 coactivator complex were mixed and incubated with glutathione beads bound to GST-Gcn4 1-134 (A) or GST alone (B). Bound fractions were analyzed by Western blotting with antibodies against Flag and GST. Relative binding values (RB) represent the percentage of the Flag-Tra1 mutant retained relative to wild-type Flag-Tra1.

Effect of TRA1 deletions on HAT subunit recruitment, histone acetylation levels, and HAT module integrity. (A to C) Strains were grown as described for Fig. and analyzed by ChIP at the indicated genes. Shown are the ChIP results obtained with Esa1-6HA and H4K8Ac (A) or Gcn5-13Myc and H3K9Ac (B and C). Histone ChIP results are shown as the ratio of acetylated to total H3 or H4 relative to the wild-type value, set at 1.0. HAT ChIP results are shown relative to the wild-type value, set at 1.0. UI, not induced. Brackets below the TRA1 mutant labels indicate the domain(s) disrupted by the deletion. P/Fc, junction between PI3K C-terminal lobe and FATc domains. (D) Relative mRNA levels from a gcn5Δ strain grown at 30°C. (E) Relative mRNA levels from an esa1 temperature-sensitive mutant strain grown at 30°C or shifted to 37°C for the indicated times before gene induction. Experiments were performed in biological duplicate, and errors bars represent standard deviations. (F and G) Western analysis of immunoprecipitated Flag-Tra1 (F) and Gcn5-13myc (G) complexes. Amounts of the immunoprecipitate (IP) loaded were normalized to load equivalent amounts of Tra1 and Gcn5, respectively. Purified complexes were analyzed by Western blotting using the following antibodies: Flag, Myc, Ada2, and Ada3. (H) Histone H3 HAT activity of Flag-Tra1 purified complexes. Equivalent amounts of Flag-Tra1 purified complexes were incubated with free histone H3 and analyzed by Western blotting using antibodies against H3K9Ac. Coomassie blue staining was used as a loading control to assess total H3 levels in each reaction mixture. Relative HAT activity levels (HA) represent the percentages of the H3K9Ac acetylation levels relative to wild-type Flag-Tra1.