Homogeneity of purified S. cerevisiae TFIID. S. cerevisiae strain YSLS18 was used for large-scale purification of HA-TAF130p-TFIID as described in Materials and Methods. Pooled anti-HA column elutes were resolved over a Mono S HR 5/5 column with a 10-ml linear gradient of potassium acetate (KOAc) from 200 to 1,000 mM in buffer A. Each fraction (5 μl, indicated across the top) was subjected to SDS-PAGE on a 10% NuPAGE gel with MOPS running buffer (Invitrogen) and visualized by staining with SYPRO Ruby. TFIID subunits are indicated on the left, and only relevant gradient fractions are shown. The elution profile for TFIID (bottom) was determined by quantitating the signals for the indicated TFIID subunits from each fraction and calculating the relative value versus the cognate TAF signal in fraction 45.

Purified S. cerevisiae TFIID efficiently mediates both basal and activated transcription. (A) Efficacy of TFIID depletion. An S. cerevisiae WCE was treated with either nonspecific (control) IgG or anti-TAF67p IgG (indicated across the top) and analyzed by immunoblotting (antibodies indicated on left) to determine the extent of TFIID depletion. (B) Quantitation of the TBP content of purified TFIID. The indicated TBP equivalents of either recombinant His₆-TBP or HA-TAF130p-TFIID-TBP were analyzed by immunoblotting. (C) Ability of purified TFIID to support specific transcription. Transcription assays were performed as described in Materials and Methods with the indicated extract (top) in the absence (−) or presence (+) of 20 ng of purified Gal4-VP16. The indicated TBP equivalent amounts were assayed for their ability to reconstitute both basal (GCN4G⁻) and activated (GAL4G⁻) transcription with the anti-TAF67p IgG-depleted WCE. The total amount of TBP (as determined by immunoblotting) contributed from each extract is indicated above the lanes.

S. cerevisiae TFIID requires TFIIA for efficient binding to the Ad2 MLP. (A) Purity of recombinant TFIIA. A 2-μg amount of purified recombinant S. cerevisiae TFIIA was subjected to SDS-PAGE and visualized by SYPRO Orange (Molecular Probes) staining. The Toa1p and Toa2p subunits of TFIIA are indicated on the right, and the mobilities of coelectrophoresed molecular mass markers (not shown) are on the left. (B) S. cerevisiae TFIID binds the Ad2 MLP specifically. In vitro footprinting reactions with the Ad2 MLP were performed as described in Materials and Methods in the absence (−) or presence (+) of recombinant TFIIA, recombinant His₆-TBP, and HA-TAF130p-TFIID, as indicated. Numbering on the left is relative to the +1 transcription start site. Brackets on the right mark the region of each lane used to generate the scanning densitometry profiles shown to the right. Traces correspond as follows: TFIIA alone (black), lane 7; TFIIA plus TBP (red), lane 3; and TFIIA plus TFIID (green), lane 6. Hypersensitive bands are marked by arrows, and a TBP equivalent-to-DNA ratio of 6:1 was used for the experiment presented. (C) Quantitation of the effect of TFIIA addition on TFIID-specific DNA binding. A titration of TBP equivalents was performed to compare the TATA binding activity of His₆-TBP versus HA-TAF130p-TFIID-TBP in the presence and absence of TFIIA. The signal of the triplet band at −30 (indicated by the bracket on the left of panel B) in the absence of either His₆-TBP or TFIID was used to determine the relative binding with increasing amounts of either His₆-TBP or TFIID. Relative values were determined from two independent experiments in which one preparation of His₆-TBP and two independent preparations of HA-TAF130p-TFIID were analyzed simultaneously. TFIID alone, black; TFIID plus TFIIA, green; TBP alone, blue; TBP plus TFIIA, red. Brackets indicate standard deviation.

S. cerevisiae TFIID interacts similarly with Ad2 MLP and natural S. cerevisiae promoters. (A, B, and C) Footprints on Ad2 MLP and RPS5 and ADH1 promoters. DNase I footprinting was performed with promoter fragments derived from the Ad2 MLP, ADH1, and RPS5 genes that had been labeled on either the top or bottom strand as indicated. Protein binding, nuclease digestion, gel fractionation, and DNA detection were performed as detailed for Fig. 3 in the presence of no added protein, TBP, TBP plus TFIIA, TFIID, or TFIID plus TFIIA, as indicated. Hypersensitive sites in the TFIID plus TFIIA reactions only are indicated by blue circles, and regions of protection are indicated by red lines. (D) Summary of the common features of TBP, TBP plus TFIIA, and TFIID plus TFIIA interactions on all three promoters. Shown are the common regions of protection (red) and nuclease hypersensitivity (blue) on all three promoters. Numbering shown is relative to the TATA box element of each gene.

Stoichiometry of TFIID subunits. Stoichiometric analyses of purified HA-TAF130p- or HA-TBP-TFIID with SYPRO Ruby and Coomassie brilliant blue R-250 stains (indicated across top) were performed as described in Materials and Methods. A representative gel and scanning densitometry profile for SYPRO Ruby-stained HA-TAF130p-TFIID are shown to the left. TFIID subunits are indicated in the first column, and the relative value for each subunit (as determined compared to TAF130p) for each stain (indicated at the top of each column) is listed across. 1, HA-TAF130p SYPRO Ruby values were averaged from independent analyses of four different preparations of HA-TAF130p-TFIID, and the standard deviation (S.D.) is listed. 2, HA-TBP-TFIID values were averaged from independent analyses of two separate preparations of HA-TBP-TFIID. TFIID subunits with an apparent stoichiometry of >1 are shaded. N.D., not determined because HA-TBP and TAF30p comigrated in the gel system used.

Self-association of TFIID subunits in vivo. (A) Blot quantitation of selected TAF-TAF in vivo interactions. A representative experiment to test the ability of TAF150p and TAF90p to self-associate is shown. Immunoprecipitations (IP) were performed with anti-Myc monoclonal antibody with WCEs prepared from diploid strains in which one of the indicated alleles (top) was Myc₁₃ tagged; − indicates the wild-type untagged diploid strain. A fraction of the input (5%) and the pellet (40%) were subjected to immunoblotting with the polyclonal antibody listed to the right to detect the protein indicated on the left. (B) Summary of self-association experimental results for all TFIID subunits. TFIID subunits with an apparent stoichiometry of >1 in Fig. 5 are shaded.

Determination of native molecular mass of purified S. cerevisiae TFIID. (A) Gel filtration sizing of TFIID. Anti-HA IgG-purified HA-TAF130p-TFIID was subjected to size fractionation on a TSK G4000SW_XL column as described in Materials and Methods. A portion of the input (In) and each fraction were subjected to immunoblotting to detect the proteins indicated (left). The void volume (V₀) and elution positions for molecular mass markers (thyroglobulin, ferritin, aldolase, and chymotrypsinogen) are shown at the top above the indicated fraction numbers (19 to 41). Note that the immunoblots for TAF150p and TAF19p were from independent experiments in which the input-to-fraction load ratio was different from that of the other subunit immunoblots. (B) Sucrose gradient sizing of TFIID. Monoclonal antibody anti-HA-purified HA-TAF130p-TFIID was subjected to rate-zonal sedimentation on a linear 10 to 30% sucrose gradient as described in Materials and Methods. A portion of the input (In) andeach fraction were subjected to immunoblotting to detect the protein indicated (left). The sedimentation positions of molecular mass markers are shown at the top. TAFs were detected by immunoblotting with appropriate antibodies; note that the blots shown for TAF150p and TAF130p used the even-numbered gradient fractions, while all the rest of the blots were prepared from the odd-numbered fractions in this particular experiment. Fraction numbers are indicated 1 to 25; top of gradient, 10% sucrose (T); and bottom of gradient (B), 30% sucrose.

Direct demonstration of TBP exchange. (A) Concentration dependence of TBP exchange within TFIID. Multiple, equal amounts of HA-TAF130p-tagged TFIID bound to protein A-Sepharose beads coated with anti-HA monoclonal antibody were generated as detailed in Materials and Methods. These aliquots of bead-bound TFIID were incubated with the indicated increasing molar excess of His₆-tagged TBP for 30 min. Unbound His₆-tagged TBP was removed by centrifugation and washing. The relative amounts of wild-type (WT) and His₆-TBP remaining bead bound to TFIID were determined by SDS-PAGE and immunoblotting with polyclonal anti-TBP IgG. This immunoblot is shown in the inset and labeled WT TBP (TFIID-endogenous TBP) and His₆-TBP (TBP exchanged into TFIID). Appropriate regions of the gel blot were separately probed with TFIID-specific antibodies (anti-TAF130p, anti-TAF65p, or anti-TAF48p IgGs). All immune complexes were detected by chemiluminescence and quantitated with a Fluor-S Bio-Rad MultiImager. Wild-type and His₆-TBP content, corrected for average TAF recovery, is plotted as percent maximal signal as a function of His₆-TBP/wild-type TBP ratio. Background subtracted and recovery-corrected wild-type TBP and His₆-TBP values, in arbitrary units, were 123 and 0, 126 and 34, 116 and 76, 124 and 136, 92 and 126, 43 and 150, 27 and 161, and 21 and 149 in the reactions with His₆-TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Average TAFp recoveries in these reactions ranged from 95 to 112%. (B) Kinetics of TBP-TFIID exchange. TBP-TFIID exchange was performed as above. Seven identical reactions were set up, each with a 10-fold molar excess of His₆-tagged TBP relative to TFIID-endogenous wild-type TBP. Samples were incubated for 0, 10, 20, 30, 45, 60, or 120 min, as indicated. Wild-type and His₆-TBP levels (upper) and TAFp content (lower) were determined by immunoblotting as in panel A. The levels of wild-type and His₆-tagged TBP in these reactions were 75 and 0, 53 and 89, 51 and 92, 49 and 87, 38 and 96, 27 and 98, and 25 and 94 arbitrary units in the reactions with His₆-TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Recovery of TAFs (lower) was in the same range as for panel A.