Mapping of Rap1p domains responsible for Rap1p-TFIID complex formation. (A) Purity of TFIID and Rap1p variants. Purified TFIID (∼2,000 ng) and ∼100 ng of the five forms of purified Rap1p (WT, ΔN, ΔC, DBD, and ΔDBD) indicated (arrows) were fractionated by SDS-PAGE and detected by Sypro Ruby staining. (B) Binding of Rap1p variants to TFIID. The five forms of Rap1p were tested for TFIID binding as detailed for the experiment of Fig. 3B; TFIID-Rap1p binding was quantitated as detailed in Materials and Methods.

Direct, high-affinity binding of Rap1p with TFIID. (A) Specificity of loading of HA₁-Taf1p-tagged TFIID beads for pull-down assay. Equivalent volumes of TFIID purified from HA-tagged Bio-Rex 70 1 M fraction (HA-TAF1) and untagged control Bio-Rex 70 1 M fraction (TAF1) were fractionated by SDS-PAGE and stained with Sypro Ruby, and TFIID subunits were visualized using a Bio-Rad FX fluorescence scanner; the image is shown. (B) Purified recombinant Rap1p interacts with TFIID. Anti-HA MAb beads were specifically loaded with TFIID (In) (A). The resulting control and TFIID-containing beads were washed extensively and incubated with a threefold mole excess (relative to TFIID) of purified E. coli-expressed Rap1p. Rap1p specifically TFIID bound was eluted with HA-peptide and detected by immunoblotting with anti-Rap1p IgG (pull-down [PD]); recovery of TFIID in the eluate was scored by probing for Taf13p.

Contribution of Rap1p domains to PIC formation and transcription in vivo. (A) Effect of Rap1p truncations on ChIP occupancy and transcription. ChIP assays were used to determine the in vivo occupancy of Rap1p and Taf1p/TFIID, TBP, and RNAP II on RPS8A, PYK1, and the UAS_RAP1-HIS3 reporter gene. Occupancy was calculated as percent enrichment in the IP over the input. Transcription (TRXN) was assessed by real time Q-RT-PCR as for Fig. 1D. Error bars indicate standard deviations. B, Assessment of the relative efficiencies of Rap1p variants in the promotion of PIC formation and transcription. Data for Taf1p, TBP, and RNAP II occupancy and transcription from panel A were normalized to Rap1p occupancy and plotted relative to WT Rap1p set to a value of 1.0; data trends are indicated by dashed lines (see text).

Model depicting Rap1p-TFIID interaction in RP gene transcription. Line 1, hypothetical RP gene with TFIID bound to the core promoter and two molecules of Rap1p bound to tandem RPG UAS_RAP1 enhancer sites. Illustrated within the trilobed (lobes A, B, and C) TFIID holocomplex (44) are the locations of TBP (red), Taf1p (yellow), and the Rap1p-interacting Tafps identified here: the two heterodimers of Taf4p-Taf12p (green) and the two molecules of Taf5p (blue). Line 2, interactions between the Rap1p DBD and C-terminal domain and the Taf12p/Taf4p and Taf5p subunits of TFIID (jagged red/yellow lines and small black curved lines). Line 3, the RP gene in the state of active transcription (wavy lines). Also shown are the additional known RP gene coregulators (i.e., Fhl1p-Ifh1p, Sfp1p, Hmo1p, Esa1p-NuA4, etc.) (light gray oval) and RNAP II plus GTFs (dark gray).

Mapping of the Rap1p binding domain of Taf12p. Serial truncations from the N or C terminus of Taf12p were expressed as GST fusion proteins and purified by glutathione-agarose chromatography. Proteins were fractionated on two identical SDS-polyacrylamide gels and blotted to PVDF membranes; one blot was directly stained with Coomassie blue (CB Stain), while the other blot was probed with Rap1p, and Taf12p-bound Rap1p was detected with anti-Rap1p IgG (Rap1p Blot). A schematic of Taf12p indicating the locations of two conserved amino acid sequence blocks, block 1 (highly conserved among Saccharomyces cerevisiae sensu stricto strains) and block 2 (the histone fold domain [HFD]), are indicated, as is a summary of the binding data (right).

Mapping of TFIID subunits capable of binding Rap1p. (A) Protein blotting test of Rap1p-Tafp interaction. Purified TFIID and recombinant His₆-tagged Tafps were fractionated by SDS-PAGE. The gel was loaded with molecular weight (MW) standards (lane 1), ∼2,000 ng purified TFIID (lanes 3 and 7), ∼250 ng E. coli-expressed His₆-Taf5p (lanes 2 and 6), ∼200 ng His₆-Taf12p (lanes 4 and 8), and ∼100 ng E. coli-expressed His₆-Taf4p (lanes 5 and 9). One part of the gel (lanes 1 to 5) was excised and stained directly with Sypro Ruby (Sypro Gel) to indicate the mobility of TFIID subunits and recombinant Tafps, while the other identical part (lanes 6 to 9) was blotted to a PVDF membrane and probed with Rap1p. Tafp-bound Rap1p was detected by immunoblotting with anti-Rap1p IgG (Bound). (B) Specificity of Rap1p binding to Taf12p. Equal amounts of E. coli-expressed His₆-Taf12p (250 ng) were fractionated by SDS-PAGE and blotted to PVDF, and individual lanes were excised from the blot and incubated with Rap1p in combination with 5- or 10-fold mole excess of either E. coli-expressed His₆-Taf12p (inset lanes 2 and 3) or purified bovine phosphodiesterase 5 (PDE5) (inset lanes 4 and 5). No exogenous competitor was added to Rap1p (inset lane 1) to determine 100%, control binding to Taf12p. PVDF-bound Taf12p was detected by staining the membrane slices with Coomassie blue (CB-Stain), while Rap1p-Taf12p complex was detected by immunoblotting with anti-Rap1p IgG (Bound) as for panel A. Rap1p binding was quantitated and graphed relative to binding in the absence of competitor. Averages of results from two replicate assays are plotted.

Rap1p drives UAS_RAP1-directed transcription in vivo. (A) Rap1p domain structure and reporter genes. The diagram illustrates the localization of Rap1p domains (top) and the various reporter genes used in this study (bottom). (B) Requirement for Rap1p binding for both TFIID recruitment and transcription in vivo. Rap1p occupancy (top), Taf1p occupancy and Taf1p/TBP occupancy ratio (middle), and in vivo transcription (bottom) of three reporter genes (UAS_RPS8A-HIS3, UAS_RAP1-HIS3, and UAS_RAP1M-HIS3) and two control genes (TAF_dep RPS8A and TAF_ind PYK1) are shown. Occupancy was calculated as percent enrichment of a gene segment in the IP relative to the input. Background occupancy was estimated as enrichment of the same proteins on the POLI gene and was subtracted. Transcription was assessed following reverse transcription of the mRNAs by quantitative real-time PCR. Error bars indicate standard deviations. (C) Dependence of RP, but not PGK, gene transcription on TFIID-Tafp function. Cells carrying the indicated TAF^ts alleles were cultured at 23°C to mid-log phase and split, and half of the culture was incubated at 23^oC while the other half was shifted to 37^oC. Total RNA was extracted after 2 h and purified, and then PGK, RPS5, and U3 RNA levels were scored by primer extension. Extension products were gel fractionated, and the sequencing gel was exposed to an imaging screen for quantitation (the scanned screen image is shown [top]; odd-numbered lanes, 23^oC cultures; even-numbered lanes, 37^oC cultures). The RPS5 RNA level was normalized to U3 RNA and graphed as percent of WT at 23^oC. (D) Effect of deletion of Rap1p domains on Rap1p-driven transcription in vivo. Steady-state mRNA levels for the three indicated genes (RPS8A, RPS5, and the UAS_RAP1-HIS3 reporter) in the strains expressing the four indicated variants of Rap1p shown were determined by Q-RT-PCR analyses as for panel B.

Rap1p and TFIID are both necessary for UAS_RAP1-driven WCE in vitro transcription. (A) Efficacy and specificity of Rap1p immunodepletion. WCEs were treated as indicated: no treatment (WCE), with protein A beads alone (Protein A), subjected to incubation but with no additions (Mock), with nonimmune rabbit IgG bound to protein A beads (Non-Imm IgG), or with anti-Rap1p IgG bound to protein A beads (α-Rap1p IgG). Equivalent amounts of each of these WCEs, along with 2 ng purified recombinant His₆-Rap1p as a positive control, were subjected to SDS-PAGE and blotted, and the blots were probed with anti-Rap1p antibody (Rap1p and His₆-Rap1p are indicated by arrows). (B) Transcription of UAS_RAP1-HIS3 is Rap1p dependent and rescued in Rap1p-depleted WCE by readdition of purified recombinant Rap1p. The control (lanes 1 to 7) and immunodepleted (lanes 8 to 11) WCEs of panel A, with either no additions (lanes 1, 2, 3, 4, 8, 12, and 13) or the addition of the indicated amounts of purified recombinant Rap1p were used for in vitro transcription experiments with a plasmid carrying the UAS_RAP1-HIS3 reporter gene as a template (Fig. 1A). Transcription of the UAS_RAP1-HIS3 reporter gene was monitored by primer extension analysis using an mRNA^HIS3-complementary probe. Primer extension of specific in vitro transcription products (mRNA^HIS3; arrow, left) comigrated with the extension product produced from authentic, in vivo mRNA^HIS3 produced from the integrated UAS_RAP1-HIS3 reporter gene (arrow, right, lane 14). Additional control reactions (lanes 12 and 13) used untreated WCE and were performed in the absence of template DNA (lane 12) or in the presence of 10 μg/ml α-amanitin (lane 13). (C) Efficacy and specificity of TFIID immunodepletion. WCE was treated as for panel A except that anti-Taf4p IgG was used as the specific antibody. Equivalent amounts of each of these WCEs, along with 2.5 ng purified TFIID as a positive control, were subjected to SDS-PAGE and blotted, and the blots were probed with antibodies recognizing the indicated proteins (Taf7p, Taf4p, and Taf13p). (D) Transcription of UAS_RAP1-HIS3 is TFIID dependent and rescued in TFIID-depleted WCE by readdition of purified TFIID but not by addition of TBP. Transcription experiments were conducted with control and anti-Taf4p IgG/TFIID-immunodepleted WCEs, as shown, in the presence and absence of the indicated amounts of TBP or TFIID. All WCEs, controls, depletions, and transcription assays were as for panels A and B. (E) Efficacy and specificity of TFIID/Rap1p double immunodepletion. WCE was treated as for panels A and C except that both bead-bound anti-Taf4p IgG and anti-Rap1p were used. Equivalent amounts of each of the resulting WCEs, along with 0.8 ng purified TFIID, 2 ng His₆-Rap1p, and 0.8 ng His₆-TBP, were run in parallel as positive controls. Proteins were subjected to SDS-PAGE and blotted, and the blots were probed with antibodies recognizing the indicated proteins (Rap1p, Taf7p, Taf4p, Taf13p, and TBP). (F) TFIID and Rap1p, but not TBP, can efficiently rescue specific transcription in doubly depleted WCE. Transcription experiments were conducted with control and anti-Taf4p/anti-Rap1p IgG doubly immunodepleted WCEs, as shown, in the presence and absence of the indicated amounts of Rap1p, TBP, or TFIID. All controls, depletions, and transcription assays were as described above. (G) The UAS_RAP1-driven HIS3 reporter gene is efficiently transcribed in the absence of the Fhl1p-Ifh1p coactivator complex. WCEs were prepared in parallel from WT (YPH252) and ΔFHL1 ΔIFH doubly deleted yeast (DR35). Equivalent amounts of protein from these two extracts (WT WCE and ΔFHL1/ΔIFH1 WCE) were tested for in vitro transcription of the UAS_RAP1-driven HIS3 reporter gene. Controls were as for panels B, D, and F. Transcription was quantitated by imaging as for Fig. 1C.