Mutations on the DNA binding surface of TBP discriminate between yeast TATA and TATA-less gene transcription.

Mutations on the DNA Binding Surface of TBP Discriminate between Yeast TATA and TATA-Less Gene Transcription

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Ivanka Kamenova, Linda Warfield and Steven Hahn Mol. Cell. Biol. 2014, 34(15):2929. DOI: 10.1128/MCB.01685-13. Published Ahead of Print 27 May 2014.

Mutations on the DNA Binding Surface of TBP Discriminate between Yeast TATA and TATA-Less Gene Transcription Ivanka Kamenova,a,b* Linda Warﬁeld,b Steven Hahnb

Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription.

T

ATA binding protein (TBP) is an essential general transcription factor (GTF) required for eukaryotic nuclear and archaeal transcription (1, 2). TBP, in combination with a subset of RNA polymerase (Pol)-specific GTFs, is a component of the promoter recognition module in all TBP-dependent transcription systems (3, 4). In the RNA Pol II system, there are two general classes of core promoters: TATA-containing promoters with a close match to the consensus sequence TATAWAWR and TATA-less promoters lacking a close match (5–10). In Saccharomyces cerevisiae, ⬃15% of Pol II promoters are TATA-containing promoters and are generally highly regulated and/or stress-inducible genes (9, 10). TBP binds the TATA element with nanomolar affinity, interacting with the minor groove and bending DNA ⬃90° (11, 12). At yeast TATA-containing promoters, the TBP-TATA interaction is important for activity, and mutation of either the TBP binding surface or the TATA sequence severely decreases transcription initiation (13–16). DNA bending by TBP is essential for subsequent binding of the GTF TFIIB, as TFIIB binds both TBP and bent DNA on either side of TATA (17, 18). Yeast Pol II promoters have also been classified by dependence on the coactivators SAGA (Spt-Ada-Gcn5-acetyltransferase) and TFIID (transcription factor IID). Approximately 10% of promoters are primarily dependent on SAGA, while close to 90% are primarily TFIID dependent (19, 20). TFIID-dependent promoters are generally TATA-less, and SAGA-dependent promoters are generally TATA-containing promoters. While these two coactivators have additional activities, they both play key roles in activator-dependent recruitment of TBP (21–24). In yeast, disruption of either SAGA or TFIID leads to reduced levels of promoter-associated TBP and preinitiation complex (PIC) formation (24–26). The roles of TBP in DNA recognition and transcription of TATA-less TFIID-dependent promoters are unclear. One widely supported model is that TFIID binding at metazoan TATA-less promoters is primarily driven by interaction of the TFIID Taf subunits with short degenerate promoter elements such as the Inr, MTE, and DPE rather than by direct TBP-DNA interactions (27).

August 2014 Volume 34 Number 15

In support of this mechanism, one or more of these alternative promoter elements are critical for transcription initiation at TATA-less promoters (8, 28). Cross-linking and other experiments show that a subset of Tafs directly interact with these elements (29–31), and Tafs can be recruited to promoters in the absence of TBP (32, 33). Also consistent with this mechanism, a mutation in human TBP that abolishes TATA-DNA binding has little or no effect on transcription in vitro from two TATA-less promoters (28). In contrast to this model, recent high-resolution mapping of TBP and TFIIB in the yeast and human genomes indicated that TBP/TFIIB cross-links adjacent to a TATA-like sequence at nearly all genes (34, 35). This finding raises the possibility that TBP directly interacts with DNA in a sequence-specific manner at these divergent sequences. For example, greater than 95% of in vivo TBP cross-linking sites are near sequences with 2 or less (yeast) or 3 or less (human) mismatches to the TATA consensus sequence. An important question is whether TBP binds TATA-like sequences with sufficient affinity and specificity to be functionally important for transcription at TATA-less genes. Initial in vitro studies showed that TBP has nanomolar affinity for several TATA sequences with one mismatch to the consensus (36). X-ray structures of TBP bound to 8 TATA-like sequences with single-base consensus mismatches showed that neither the structure of TBP nor the DNA trajectory is altered in these complexes (37). Because TBP recognizes bases in the DNA minor groove, A/T substitution

Received 19 December 2013 Returned for modification 20 January 2014 Accepted 18 May 2014 Published ahead of print 27 May 2014 Address correspondence to Steven Hahn, [email protected]. * Present address: Ivanka Kamenova, IGBMC, Illkirch, France. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/MCB.01685-13

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Program in Molecular and Cellular Biology, University of Washington,a and Division of Basic Sciences, Fred Hutchinson Cancer Research Center,b Seattle, Washington, USA

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TABLE 1 Yeast strains used in this work Genotype

Reference

SHY737 YSB373 taf40-3100 YSB373 SHY626 SHY763 SHY851

mat␣ ade2⌬::hisG his3⌬200 leu2⌬0 lys2⌬0 met15⌬0 trp1⌬63 ura3⌬0 spt15⌬::KanMx (ars cen URA3 SPT15) mata ura3-52 leu2 trp1⌬63 his3⌬200 taf11⌬::LEU2/pRS313-taf11-3100 (ars cen HIS3 taf11-3100 ts) mata ura3-52 leu2 trp1⌬63 his3⌬200 taf11⌬::LEU2/pRS313-TAF11 (ars cen HIS3 TAF11) mat␣ ade2⌬::hisG his3⌬200 leu2⌬0 lys2⌬0 met15⌬0 trp1⌬63 ura3⌬0 TAF1-(1⫻Flag)-TAP tag::TRP1 mata ura3-52 trp1 lys2-801 leu2⌬1 his3⌬200 pep4::HIS3 prb1⌬1.6R can1 TAF1-Flag3::KanMx TAF3-Flag3::HPH mata ade2⌬::hisG his3⌬200 leu2⌬0 lys2⌬0 met15⌬0 trp1⌬63 ura3⌬0 spt7⌬::HPH

24 64 64 24 67 This work

is tolerated at many individual positions. In addition, cavities in the TBP binding surface allow single C or G substitutions at TATA motif position 3 or 6, although with significant reduction in TBP affinity (37). Finally, since TBP binds TATA using an induced fit mechanism, the bendability of DNA likely affects TBP binding affinity; e.g., DNA with long A stretches does not efficiently bind TBP. Although early biochemical studies suggested that TATA variants could alter the DNA trajectory in the TBP-DNA complex (38, 39), recent single-molecule experiments agree with the structural studies that DNA trajectory is not affected when TBP is bound to single-base variants (40). However, none of these biochemical studies has successfully measured TBP binding to TATA variants containing two or three mismatches to the consensus, which encompass 37 to 85% of the TATA-like promoter sequences in yeast and human (34, 35). Here we have investigated whether sequence-specific TBPDNA interaction is important for transcription of S. cerevisiae RPS5, a well-characterized TATA-less, TFIID-dependent promoter (41–43). Apart from TATA, the GA element (GAAAA) (10) and the conserved sequence at the transcription start site (44, 45), yeast promoter specificity elements equivalent to the human TFIIB binding sites (BREu and BREd) (46) and metazoan Taf binding sites (Inr, MTE, and DPE) have not been identified (47). Thus, it is not clear how promoter specificity can be generated at yeast TATA-less promoters if TBP cannot directly recognize DNA in a sequence-specific manner. While we found that TBP binds three separate regions of the RPS5 promoter, our functional studies suggest that this binding is not important for RPS5 transcription, as mutations on the TBP binding surface that reduce or abolish RPS5 binding have little effect on RPS5 transcription. Our combined results suggest that sequence-specific TBP binding is not required at yeast TATA-less promoters and that promoter specificity must be generated by alternative mechanisms. MATERIALS AND METHODS Yeast strains and culture. All strains used for viability assays and quantitative PCR (qPCR) analyses are derivatives of the TBP shuffle strain SHY737 (Table 1). Strains were constructed by lithium acetate transformation followed by shuffling of the TBP-containing plasmid by selection on 5-fluoroorotic acid plates. The yeast strains were grown in either rich medium (yeast extract-peptone-dextrose [YPD] with 3% dextrose) or glucose complete (GC) medium with 2% dextrose and lacking the appropriate amino acids as noted. For induction of Gcn4-dependent genes, cells were grown in GC medium lacking isoleucine and valine and induced with 0.5 ␮g/ml sulfometuron methyl (SM) dissolved in dimethyl sulfoxide (DMSO) for 1 h. Experiments with strains containing TBP temperature-sensitive (ts) mutations were carried out by growing cells to an optical density at 600 nm (OD600) of ⬃0.8 at 25°C and shifting cells to 37°C for 30 min, followed by growth for 1 h with 0.5 ␮g/ml SM at 37°C. Viability assays were performed using GC plates lacking isoleucine and valine with

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or without 3 ␮g/ml SM. Fivefold serial dilutions were spotted, and plates were incubated at 30°C for 2 days before being photographed, with the exception of SM plates which were photographed after 3 days. mRNA quantitation by RT-qPCR. The total RNA extraction and reverse transcription-quantitative PCR (RT-qPCR) procedures were performed as described previously (48) except that the Pol I transcript RDN18-1 was used as a reference gene for normalization and that random hexamer plus oligo(dT) was used during cDNA synthesis. WCE preparation. Twelve liters of cells was grown in YPAD (1% [wt/vol] yeast extract, 2% [wt/vol] peptone, and 3% [wt/vol] dextrose, supplemented with 0.004% [wt/vol] adenine), to an OD600 of ⬃2 to 3. The cells were washed once with distilled water, and the cell pellets were transferred to 50-ml conical tubes, frozen in liquid nitrogen, and kept at ⫺80°C until further processing. Cell pellets were thawed in a room temperature water bath, transferred to ice, and resuspended in ⬃1 ml cold lysis buffer supplemented with dithiothreitol (DTT) and protease and phosphatase inhibitors per gram of cell pellet. The lysis buffer composition was as follows: 200 mM Tris-acetate (pH 7.9), 390 mM ammonium sulfate, 20% glycerol, and 1 mM EDTA. DTT (1 mM), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM benzamidine, 2.5 ␮g/ml leupeptin, 1.5 ␮g/ml pepstatin, 10 ␮g/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK), 10 ␮g/ml N␣-p-tosyl-L-lysine chloromethyl ketone (TLCK), 0.2 mM NaF, 0.2 mM ␤-glycerophosphate, 0.2 mM activated sodium orthovanadate, and 300 nM trichostatin A were added immediately before use. The resuspended cell pellets were placed in the chilled chamber of a bead beater (BioSpec Products) filled halfway with chilled 0.5-mm glass beads. The cells were lysed with 12 30-s pulses and 2-min breaks and kept cold throughout the procedure. Crude extracts were collected, and additional proteins were extracted by washing the beads with 50 ml cold lysis buffer. The combined crude extract was clarified by centrifugation in an FL14 rotor (30 min at 16,000 ⫻ g) to remove cell debris and then spun at 4°C for 3 h at 200,000 ⫻ g in a Beckman 50.2 Ti rotor. The clarified extract was transferred to a glass beaker, solid ammonium sulfate was added to 0.337 g/ml, and the mixture was stirred for 30 min at 4°C. The precipitated protein was collected by centrifugation (15 min at 200,000 ⫻ g in a Beckman 50.2 Ti rotor at 4°C) and resuspended slowly in a minimal amount (⬃1 ml/liter culture) of cold dialysis buffer (20 mM HEPES [pH 7.9], 20% glycerol, 10 mM magnesium sulfate, 10 mM EGTA, 5 mM DTT, with protease and phosphatase inhibitors as listed above). The whole-cell extract (WCE) was dialyzed against three 1-liter changes of dialysis buffer until the conductivity of the extract diluted at 1:200 was less than 100 ␮S/cm. The resulting whole-cell extract was clarified at 27,000 ⫻ g and stored at ⫺80°C. For the preparation of TFIID-depleted whole-cell extracts, yeast strain SHY343 (taf11 ts) and the isogenic wild-type (WT) strain YSB373 were grown at 25°C until an OD600 of 2 to 3 was reached. Cells were resuspended in the equivalent amount of 37°C YPAD medium and incubated in a 37°C shaker for 1 h. The remainder of the whole-cell extract procedure was as described above. TFIID purification. TFIID was purified from yeast strain SHY626 expressing tandem affinity purification (TAP)-tagged Taf13. Twelve liters of culture was grown in YPD (3% dextrose) to an OD600 of ⬃2.0 and harvested by centrifugation. The cell pellet was washed in 200 ml cold TAP extraction buffer (40 mM HEPES [pH 7.5], 10% glycerol, 150 mM NaCl, 0.1% Tween 20) without protease inhibitors or DTT, and resuspended in



Strain

TBP at TATA-Less Promoters


ampicillin until an OD600 of ⬃0.7 was reached. Expression was induced with 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) for 3 h at 37°C. The cells were collected by centrifugation, washed in 50 ml lysis buffer (30 mM Tris-HCl [pH 7.5], 500 mM KCl, 40 mM imidazole, 10% glycerol), resuspended in 30 ml lysis buffer, quick-frozen in liquid nitrogen, and stored at ⫺70°C until further use. DTT and PMSF were added to the thawed pellet to a final concentration of 1 mM each, and cells were treated with 0.5 mg/ml lysozyme (Sigma) for 30 min on ice and then lysed by sonication. The resulting lysates were clarified by centrifugation and incubated with 2 ml equilibrated nickel-Sepharose beads (GE Healthcare) for 1 h at 4°C. After the beads were collected, they were washed four times with lysis buffer (10 min at 4°C per wash), and the bound protein was eluted four times in 4-ml fractions (the elution buffer consisted of 30 mM Tris [pH 7.5], 500 mM KCl, 500 mM imidazole, and 10% glycerol). After the appropriate fractions were pooled, the protein was dialyzed into SUMO cleavage buffer (30 mM Tris-HCl [pH 7.5], 300 mM KCl, 10% glycerol) over the course of 3 h at 4°C with three 1-liter buffer changes. The dialyzed protein was spun to remove precipitated material, recombinant SUMO protease was added to a final concentration of ⬃2.2 ␮g/ml, and digestion was carried out for 3 h at 4°C. The SUMO tag and protease were removed by incubating the cleaved protein with nickel-Sepharose beads for 1 h at 4°C. The salt concentration of the digested protein sample was reduced to 150 mM by dialysis, and the protein was purified on a Source 15S column equilibrated in 7.5% buffer B (20 mM Tris-HCl [pH 7.8], 2 M KCl, 10% glycerol) and eluted with a gradient from 150 mM to 500 mM KCl. Fractions were analyzed by Coomassie blue staining, pooled, and concentrated (if needed) in Amicon Ultra or YM-10 concentrators with 10,000 (10K)-molecular-weight cutoff (Millipore). DTT (1 mM) was added to all buffers immediately before use. In vitro transcription and primer extension. In vitro transcription reaction mixtures (25 ␮l) contained 20 mM HEPES (pH 7.6), 100 mM potassium acetate, 1 mM EDTA, 5 mM magnesium acetate, 3 mM DTT, 0.4 mM each nucleoside triphosphate (NTP), 38 mM creatine phosphate, 0.03 unit creatine phosphokinase, and 4 units RNase Out (Invitrogen). One microliter of supercoiled plasmid DNA template (150 ng for pSH515, 300 ng for all TATA-less promoter constructs) was added to each reaction mixture, along with 24 ng recombinant Gal4-VP16 activator, and the activator and DNA were allowed to bind for 10 min at room temperature. Transcription was initiated by the addition of 150 to 250 ␮g whole-cell extract or 60 to 90 ␮g nuclear extract and allowed to proceed for 40 min at room temperature. Stop mix (180 ␮l) consisting of 100 mM sodium acetate, 10 mM EDTA, 0.5% SDS, and 17 ␮g/ml tRNA (Sigma) was added to stop the reaction. Samples were assayed by primer extension as described previously (50). EMSAs. Binding assays (20 ␮l) contained 20 mM Tris-HCl (pH 8.0), 60 mM KCl, 5 mM MgCl2, 4% glycerol, ⬃6,000 cpm double-stranded promoter DNA probe (5= end labeled with 32P by PCR and gel purified using a Qiagen gel extraction kit), 0.5% Brij 58, 0.3 ␮g/ml bovine serum albumin (BSA) (NEB), 0.125 ␮g poly(dG-dC), and proteins diluted in protein dilution buffer (20 mM Tris [pH 7.9], 150 mM KCl, 1 mM DTT, 10% glycerol, 50 ␮g/ml BSA). The proteins were added last, and the reaction mixtures were incubated for 30 to 40 min at room temperature before they were loaded onto 6% native acrylamide gels. 1⫻ TGOE (25 mM Tris [pH 8.3], 0.19 M glycine) plus 0.2 mM MgOAc in the gel and running buffer was used for the RPS5 assays, and 1⫻ TGOE plus 1 mM EDTA and 5 mM MgOAc was used for the HIS4 assays. Electrophoresis was carried out for 1 h at 4°C. Gels were dried and analyzed by a PhosphorImager. For competition electrophoretic mobility shift assays (EMSAs), proteins and competitor DNAs were incubated for 10 min before the addition of 32Plabeled DNA for 20 min. DNase I footprint assays. DNase I analysis of protein-DNA interactions followed a standard protocol (51) with several modifications. The conditions for protein-DNA binding were identical to the ones described above in the gel mobility shift assays except ⬃20,000 cpm of end-labeled double-stranded DNA probe was used. After a 30-min incubation of DNA

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150 ml cold TAP extraction buffer with DTT and protease inhibitors. Unless otherwise specified, DTT (final concentration of 1 mM) and protease inhibitors (49) were added to all buffers immediately before use. Cells were lysed in a bead beater (BioSpec Products) as described above for WCE preparation, and the supernatant was clarified by centrifugation in a FL14 rotor (⬃15,000 ⫻ g for 30 min at 4°C). The clarified supernatant was subjected to ultracentrifugation in a fixed-angle 50.2 Ti rotor (Beckman) at 150,000 ⫻ g for 90 min at 4°C. The supernatant was either quick-frozen in liquid nitrogen and stored at ⫺70°C until further processing or directly used for IgG purification. IgG-Sepharose beads (GE Healthcare) (2.5 ml) were equilibrated in TAP extraction buffer and added to the whole-cell extract and incubated on a roller at 4°C overnight. The beads were collected by centrifugation and transferred at 4°C to an Econo-Column (BioRad) (5/8-in. diameter, 5-in.-long column). After the depleted extract was drained, the IgG beads were washed five times with 10 ml TAP extraction buffer and then washed four times with 10 ml TEV cleavage buffer (10 mM Tris [pH 8], 150 mM NaCl, 0.1% NP-40, 0.5 mM EDTA, 10% glycerol). The IgG beads were resuspended in 4 ml TEV cleavage buffer and transferred to a 15 ml screw-cap tube. Tobacco etch virus (TEV) protease (25 ␮g) was added, and the slurry was incubated overnight on a roller at 4°C. Calmodulin-agarose beads (Stratagene) (1 ml) were equilibrated by washing the beads three times with 14 ml calmodulin binding buffer (15 mM HEPES [pH 8], 1 mM magnesium acetate [MgOAc], 1 mM imidazole, 2 mM CaCl2, 0.1% NP-40, 10% glycerol, 150 mM NaCl). The TEVtreated IgG resin was transferred to the Econo-Column described above, and the supernatant was collected in a screw-cap tube. The column was washed with 3 column volumes (⬃7.5 ml total) of calmodulin binding buffer, and the wash was combined with the supernatant obtained from TEV cleavage. CaCl2 was added to the combined flowthrough and wash fractions at a final concentration of 2 mM, and the protein was bound to 1 ml equilibrated calmodulin-agarose beads. After a 4-h incubation at 4°C (or overnight), the beads were washed twice with 14 ml calmodulin binding buffer, incubating each wash ⬃5 min at 4°C. The beads were then washed three times with 14 ml each calmodulin wash buffer and resuspended in 3 ml calmodulin wash buffer (identical to calmodulin binding buffer, but detergent and protease inhibitors can be omitted if needed). The resultant slurry was transferred to a 10-ml disposable column (BioRad) with the flow rate adjusted to ⬃0.5 ml/min. The bound TFIID complex was eluted by sequential addition of 11 0.5-ml fractions of room temperature calmodulin elution buffer (15 mM HEPES [pH 8], 1 mM MgOAc, 1 mM imidazole [high purity], 2 mM EGTA, 10% glycerol, 150 mM NaCl). The elution procedure was carried out at room temperature. Fractions were analyzed by silver staining, and the desired peak fractions were pooled. For preparation of crude TFIID via Flag epitope affinity purification, ⬃500 ␮l of whole-cell extract prepared from yeast strain SHY763 expressing 3⫻Flag-tagged Taf1 and Taf3 was dialyzed for 1 h at 4°C against WCE dialysis buffer (described above in “WCE preparation”) supplemented with protease inhibitors to remove DTT. Flag-M2-agarose bead slurry (Sigma) (120 ␮l) equilibrated in dialysis buffer was added, and the extracts were incubated for 1.5 h at 4°C. The depleted WCE was added to a fresh batch of M2 beads (120 ␮l), and the 1.5-h incubation was repeated. The beads from the two consecutive depletions were combined and washed twice in 1 ml dialysis buffer supplemented with protease inhibitors. Bound TFIID was eluted by incubation with 120 ␮l of 3⫻Flag peptide (Sigma) at 0.25 ␮g/␮l for 20 min at room temperature with nutation. The elution was repeated once, and the eluates were frozen on dry ice and stored at ⫺70°C. TBP purification. Full-length yeast TBP or yeast TBP core (TBPc) (residues 61 to 240) was expressed as N-terminal SUMO (small ubiquitinlike modifier) fusion proteins (Invitrogen) from pIK8 (6⫻HIS-SUMOTBP in pET21a) and pIK12 (6⫻HIS-SUMO-⌬2-60 TBP in pET21a) in Escherichia coli strain BL21(DE3)RIL. Two liters of cells (for WT) or 4 to 12 liters (for mutants) was grown at 37°C in 2⫻ YT medium (10 g NaCl, 16 g tryptone, 10 g yeast extract per liter) supplemented with 50 ␮g/ml

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and proteins, 2 ␮l of DNase I (USB; ranging from undiluted to 8-fold diluted enzyme) was added, and the reaction was stopped after incubation for 1 min at room temperature by the addition of 160 ␮l stop mix (20 mM EDTA, 31 ␮g/ml tRNA). The reaction products were ethanol precipitated, the pellets were resuspended in formamide gel loading buffer (80% deionized formamide, 30 mM EDTA, 0.04% bromophenol blue, 0.04% xylene cyanol), and analyzed on a denaturing acrylamide-urea sequencing gel. The gels were analyzed by a PhosphorImager.

RESULTS

Many mutations in the TBP DNA binding surface support yeast growth. To address whether the sequence-specific DNA binding activity of TBP is used at TATA-less promoters, we first created a series of mutations on the DNA binding surface of S. cerevisiae TBP and assessed the effects of these mutations in vivo and in vitro. We selected 11 conserved residues for mutation based

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on their positions in the TBP-DNA interface and, in some instances, known in vivo phenotypes (Fig. 1A) (11, 12, 16, 52). All mutated residues except T112 and K218 directly contact either the bases or the backbone of TATA DNA. When TBP is not bound to TATA, its DNA binding surface can also interact with Mot1, an ATP-dependent regulator of TBP binding (53), or the TAND1 domain of the TFIID subunit Taf1 (54, 55), or dimerize with another molecule of TBP (56). As detailed below (see Discussion), the phenotypes of our TBP mutations are distinct from those caused by mutations within Mot1, the Taf1 TAND1 domain, and disruption of TBP dimerization, so we believe that our TBP mutations exert their primary effects due to altered TBP-DNA binding. Most selected TBP residues were changed to alanine, and these included Phe 116 and Phe 207, which are responsible for base



FIG 1 TBP mutations in the protein-DNA interface. (A) Amino acid substitutions generated on the DNA binding surface of yeast TBP. Mutated residues are shown in cyan. (B) Recombinant TBP core (TBPc) proteins. Proteins were purified as described in Materials and Methods and visualized by SDS-PAGE and Coomassie blue staining. The positions of protein standard markers (M) (in kDa) are indicated to the left of the gel. (C) Electrophoretic mobility shift assay (EMSA) with radiolabeled TATA-containing HIS4 promoter DNA probe. Wild-type and mutant recombinant TBP core (residues 61 to 240) was used at concentrations ranging from 1.25 to 5 nM (indicated by the height of the triangle above the lane).


mutants assessed by a spot test assay. Fivefold serial dilutions of yeast were spotted on plates containing glucose complete (GC) medium without Ile and Val with 3 ␮g/ml sulfometuron methyl (SM) or without SM. (B) Summary of yeast growth phenotypes in panel A. Symbols: ⫺ to ⫹⫹⫹⫹, cell growth rate ranging from no growth to wild-type growth.

stacking interactions that result in kinking and bending of TATA DNA. Two nonalanine mutations, T112K and S118L, were also generated, based on previous phenotypic analysis. Human TBP mutant T120K (equivalent to yeast T112K) was found to selectively inhibit transcription from a TATA-containing promoter (57), while S118L is defective in response to several acidic activators (52). An electrophoretic mobility shift assay was used to demonstrate that recombinant T112K, F116A, and V71A TBP mutants were defective in DNA binding at the HIS4 TATA-containing promoter (Fig. 1B and C). For this assay, the TBP mutants were tested for DNA binding in the context of the conserved TBP core (TBPc) domain (residues 61 to 240), as these mutant proteins were more soluble and stable in this context. Most of the other recombinant TBP mutant proteins were insoluble both as full-length TBP and as TBPc. Plasmids containing individual mutations in the TBP DNA binding surface were tested for their ability to support yeast growth using a plasmid shuffle assay. Surprisingly, the majority of TBP derivatives gave viable yeast, with the exception of mutation T112K (Fig. 2A and B). The mutant TBPs displayed a range of


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FIG 2 Growth phenotypes of yeast TBP mutants. (A) Growth of viable TBP

growth phenotypes on synthetic complete glucose medium: the R105A TBP mutant grew slowly under all conditions tested, the V71A, V161A, and L205A TBP mutants had mild growth phenotypes, and the rest were indistinguishable from strains with wildtype TBP. These results demonstrate that normal TBP DNA binding activity is not required under optimal growth conditions and is consistent with the hypothesis that TBP sequence-specific DNA binding activity is not required for transcription of TATA-less genes. As a first test of whether the TBP DNA binding surface mutations give rise to defects in expression of TATA-containing stress response genes, we assayed the mutants for growth in the amino acid biosynthesis inhibitor sulfometuron methyl (SM) that is used to mimic amino acid starvation and induce a cluster of genes regulated by the activator Gcn4 (58). The inability to grow in the presence of SM is an indicator of failure to induce genes in the Gcn4 pathway. Growth assays revealed that all mutants except V203A and K218A TBP mutants were sensitive to SM, indicating that most have a defect in induction of genes in the Gcn4 pathway. These results are consistent with previous findings that a subset of TBP DNA binding mutants are specifically defective for activated transcription (52, 59). Disruption of the TBP DNA binding surface reduces transcription at TATA-containing, not TATA-less, promoters. To measure activity of the TBP mutants at both TATA-containing and TATA-less genes, we generated strains containing both the TBP temperature-sensitive allele T111I (60) and a second copy of either wild-type TBP or the various TBP DNA binding surface mutants. For assay of Gcn4-dependent gene expression, cells were shifted to 37°C for 30 min to inactivate ts TBP. Next, SM was added to induce translation of Gcn4, and cells were grown an additional 1 h at 37°C to allow transcription of the Gcn4-dependent genes. For measuring expression of the TATA-less ribosomal protein genes, cells were heat shocked for a total of 90 min at 37°C without SM addition. mRNA expression was assayed by RT-qPCR at two TATAcontaining genes, HIS4 and SNZ1, and two TATA-less genes, RPS5 and RPL5 (Fig. 3A and B). The experimental design allowed us to assay transcriptional defects using both viable and inviable TBP derivatives. Nearly all TBP mutant proteins were expressed at levels similar to wild-type TBP irrespective of heat shock or SM addition (Fig. 3C). The two exceptions are T112K and V161A TBP, which were expressed at a lower level under all conditions. After heat shock and SM induction of cells containing wildtype TBP, the levels of expression of HIS4 and SNZ1 were induced 3.1- and 8.4-fold (Fig. 3A, WT versus WT -SM). In contrast, in strains containing only the ts TBP allele, the induced level of HIS4 and SNZ1 expression was reduced 2.6- and 5.2-fold, respectively, while RPS5 and RPL5 transcription was reduced ⬃2.6-fold (Fig. 3A and B, WT versus vector). Most TBP mutants, except for V203A and K218A TBP, failed to rescue expression (ⱖ65% of WT) of SNZ1 and HIS4. The V71A, R105A, T112K, R196A, F207A, and V161A TBP mutants all gave less than 30% wild-type HIS4 and SNZ1 transcription, which is similar to expression levels in the strain containing only the ts TBP allele. In contrast to these results, expression levels of the TATA-less ribosomal protein genes RPS5 and RPL5 were rescued by most of the TBP DNA binding surface mutants, with nearly all supporting ⱖ80% of wild-type RPS5 and RPL5 transcription (Fig. 3B). The two exceptions are the R105A and T112K TBP mutants, which only partially complemented. Our combined results showed that the V71A,

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levels from the Gcn4-dependent, TATA-containing genes HIS4 and SNZ1. All strains contained both the TBP T111I ts mutation and the indicated TBPcontaining plasmid. The ts TBP was inactivated by 30-min incubation at 37°C followed by SM induction for 60 min at 37°C. HIS4 and SNZ1 mRNA levels were normalized to RDN18-1 RNA and are expressed as a percentage of the WT level where the wild-type culture was induced with SM under the same growth conditions. Assays were done in triplicate. and error bars represent the standard errors of the means (SEMs) from two biological replicates. (B) RT-qPCR analysis of transcription from the TATA-less ribosomal protein genes RPS5 and RPL5 where the TBP T111I was inactivated for 90 min as described above for panel A but without SM addition. RPS5 and RPL5 mRNA levels were normalized to RDN18-1 RNA. Error bars represent the SEMs from two biological replicates. (C) Western blot analysis of TBP derivatives in the strains used in panels A and B. Samples were collected before heat treatment (at 25°C) and at the time of harvesting cells for expression analysis. TBP was visualized via a 3⫻Flag tag on the N terminus. The temperature (T) is shown at the top of the panel.

R196A, F207A, and V161A TBP mutants were the most affected for transcription at two TATA-containing genes while having only minor defects in expression at the two TATA-less genes. These results are consistent with the hypothesis that the sequence-specific DNA binding activity of TBP is not important for transcription from TATA-less promoters, and they explain why certain TBP DNA binding defective mutants appear to have defects in transcription promoted by several acidic activation domains (52, 59). TBP, but not an intact DNA binding surface, is required for in vitro transcription from RPS5. To examine the mechanism of

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the TBP DNA binding requirement in more detail, we developed an in vitro transcription system using the TATA-less RPS5 core promoter fused to an upstream Gal4 DNA binding site in place of the RPS5 upstream activating sequence (UAS) (Fig. 4A). Previous studies showed that the acidic activators Gal4-VP16 and Gal4 can activate RPS5 transcription in vivo in a TFIID-dependent manner (61, 62). In vitro transcription reactions performed with supercoiled plasmid template, yeast whole-cell extract, and recombinant Gal4-VP16 activator yielded transcripts originating from the transcription start site (TSS) region used in vivo (Fig. 4B). Mapping the in vitro TSSs showed that the second-most-upstream TSS



FIG 3 Many mutations in the TBP DNA binding surface selectively affect expression from TATA-containing promoters in vivo. (A) RT-qPCR analysis of mRNA


corresponds to the major in vivo TSS, although most initiation occurs in a region up to 27 bp downstream from position ⫹1 (Fig. 4C). It was recently proposed that the ⫹1 nucleosome might function to limit TSS scanning at TATA-less promoters (34), and our results are consistent with this proposal, as these reactions were conducted in the absence of nucleosomes. Addition of ␣-amanitin to the in vitro reaction mixtures inhibited transcription, demonstrating that the observed transcripts were due to Pol II activity (Fig. 4B, lane 5). To investigate transcriptional defects due to mutations in the TBP DNA binding surface, we first established a TBP-depleted in vitro system. Whole-cell extract from a strain containing the TBP ts mutation I143N, which is deficient for Pol I, II, and III transcription in vitro (49, 63), was defective for both RPS5 and HIS4 transcription in our in vitro system (Fig. 5, lanes 2, 10, and 18). Supplementing reaction mixtures with recombinant wild-type TBPc restored transcription as expected (Fig. 5, lanes 3 and 4, 11 to 13, and 19 to 21). We next assayed the ability of recombinant TBP derivatives to rescue transcription in the TBP-deficient extract. Supplementing this extract with V71A or F116A TBPc restored activity to or nearly equivalent to wild-type TBPc at RPS5 but poorly complemented transcription at the HIS4 promoter. Similarly, TBP T112K was ⬃50% active at RPS5 but was inactive at HIS4 (Fig. 5, lanes 5 to 8, 14 to 16, and 22 to 30). Thus, both in vivo and in vitro, the sequence-specific DNA binding activity of TBP appears less important on TATA-less promoters than on TATAcontaining promoters. Coactivator requirements of the RPS5 core promoter are retained in vitro. Coactivator requirements for the RPS5 promoter have been studied extensively in vivo, showing that TFIID-specific Tafs are enriched on this promoter and required for transcription,


while SAGA inactivation has little or no effect (20, 25, 33, 61). To test the TFIID dependence of the in vitro system, we used a yeast strain harboring a TAF11 temperature-sensitive mutation (64). Shifting this mutant to the restrictive temperature results in severe defects in the expression of TFIID-dependent genes. Whole-cell extracts prepared from the taf11 ts strain grown at 37°C were deficient in transcription from the RPS5 promoter (Fig. 6A, lane 2). Addition of either TAP-tagged purified TFIID (TAP-TFIID) or a less highly purified Flag-tagged TFIID (Flag-TFIID) restored transcription to greater than or equal to wild-type levels (Fig. 6A, lanes 3 to 5), while addition of recombinant TBP or TAP-tagged purified SAGA did not (Fig. 6A, lanes 6 to 11), demonstrating that in vitro transcription of RPS5 is TFIID dependent. To test for SAGA dependence of the in vitro system, whole-cell extracts were made from a strain containing a deletion of SPT7, a key SAGA subunit (65, 66). We observed that the ⌬spt7 extract was functional for transcription of the RPS5 promoter and that addition of purified SAGA did not further stimulate transcription (Fig. 6B). In contrast, depletion of SAGA results in loss of activator-dependent transcription from the TATA-containing HIS4 promoter that can be restored by addition of purified SAGA (67). Collectively, our findings indicate that our in vitro system has the same coactivator requirements for RPS5 transcription as observed in vivo. TBP can bind to the TATA-less RPS5 promoter, but these specific sites are not important for transcription. TBP can bind specifically to DNAs with a one-base mismatch to the TATA consensus with an affinity up to several orders of magnitude greater than nonspecific DNA (36, 37). However, the ability of TBP to bind naturally occurring TATA-less promoter DNA has not been studied in detail. We first tested the ability of the RPS5 promoter

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FIG 4 In vitro transcription of the TATA-less RPS5 promoter. (A) Schematic of the RPS5 core promoter construct used for in vitro transcription assays. The RPS5 core promoter sequence (positions ⫺103 to ⫹106 relative to the start of transcription) was fused to an upstream Gal4 binding site. Transcription was visualized by reverse transcription (RT) with a radiolabeled or fluorescently labeled lacI primer whose position is shown. (B) In vitro transcription from the RPS5 promoter. A 32P-labeled primer was used in RT. The amounts of yeast whole-cell extract (WCE) are shown above the gel, and recombinant Gal4-VP16 (24 ng) and ␣-amanitin are added as indicated. (C) Mapping the transcription start sites from RPS5 in vitro transcription. (Top) Primer extension products from RPS5 in vitro transcription were resolved on a high-resolution DNA sequencing gel. The bracket denotes the transcription TSSs, and the major in vivo TSS is labeled ⫹1. Sequencing reactions of the RPS5 promoter template used as size markers are also shown. (Bottom) In vitro TSSs are marked with asterisks on the RPS5 coding strand, and the major in vivo start site, labeled ⫹1, is underlined.

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reaction mixtures contained either WT extract (lanes 1, 9, and 17) or TBP I143N extract defective in TBP activity in vitro. The reaction mixtures also contained Gal4-VP16 and the indicated TBP derivatives (24 to 72 ng). RNA was visualized by RT using a fluorescently labeled lacI primer. Promoters used were either the RPS5 promoter from Fig. 4 or the HIS4 promoter from pSH515 (49). NE, nuclear extract.

to compete for TBP binding at the HIS4 TATA-containing promoter (Fig. 7A). In a gel mobility shift assay with a labeled HIS4 promoter probe, addition of cold unlabeled HIS4 or RPS5 competitor but not HIS4 with a mutated TATA box significantly reduced levels of TBPc bound to the HIS4 promoter. For example, a 50-fold molar excess of RPS5 promoter DNA decreased TBP-HIS4 binding to near undetectable levels. Importantly, RPS5 was a more effective competitor than HIS4, showing that TBP binds RPS5 with a higher affinity than HIS4. Competition experiments with two other TATA-less promoters, RPL9a and RPL25, revealed similar results, with both promoters competing for TBPc at least as well or better than HIS4 (Fig. 7B). These findings collectively led to the conclusion that TBP is capable of binding to at least some TATA-less promoters with relatively high affinity. To extend these results, we directly examined TBP binding to RPS5 using a mobility shift assay (Fig. 7C). TBPc, but not fulllength TBP, bound to RPS5, and binding of both forms of wildtype TBP was enhanced upon addition of TFIIA, a GTF known to stabilize TBP-DNA binding (68, 69). TFIIA also stabilized both forms of TBP at HIS4 as expected. We next tested whether three TBP derivatives that failed to bind HIS4 but stimulated transcription at RPS5 could bind the RPS5 promoter (Fig. 7D). As expected, binding of TBPc T112K, F116A, or V71A was nearly undetectable at RPS5. Upon addition of TFIIA, binding of TBP F116A and V71A was detectable but at a low level compared to wild-type TBP. Taken together, our results suggest that even though TBP can bind several TATA-less promoters, this binding is not critical for transcription initiation, since three mutations that eliminate or significantly reduce TBP DNA binding at RPS5 can promote transcription from RPS5. A DNase footprinting assay was next used to identify the loca-

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tions of TBP bound to RPS5 (Fig. 8). We found that TBPc binds RPS5 at three distinct sites with the two most prominent binding sites at positions ⫺74 to ⫺61 (footprint 1 [FP1]) and ⫺40 to ⫺16 (FP2). A third weaker footprint (FP3) is centered over the TSS at approximately positions ⫺8 to ⫹3. FP1 is centered over the sequence TATCAATA, which is not expected to bind TBP using the normal TBP-TATA interface, as there should be no room to accommodate the exocyclic NH2 group from guanine in the TBP DNA binding surface (37). FP2 is ⬃30 bp in length, about twice as large as a TBP footprint on a consensus TATA sequence (70). This expanded footprint has been observed on at least several nonconsensus TATAs (36) and may represent either binding of multiple TBP molecules or an unconventional binding mechanism. Binding of TBPc to RPS5 was observed both in the presence and absence of TFIIA, and the amount of TBP necessary for binding was nearly equivalent to the amount needed to footprint the HIS4 TATA box (Fig. 8), although at saturation, the HIS4 footprint showed more complete protection from DNase. An interesting question is whether any of these TBP binding sites correspond to the sites of TBP binding in vivo. The in vivo binding sites for yeast TBP, TFIIB, Pol II, and other general transcription factors have been mapped genome-wide at high resolution using chromatin immunoprecipitation using exonuclease (ChIP-exo) (34; H. S. Rhee and F. Pugh, personal communication). These data show a broad primary peak of TFIIB-DNA crosslinking on the RPS5 nontemplate strand between positions ⫺28 and ⫺2, suggesting a single predominant (but perhaps fuzzy) preinitiation complex (PIC) location. However, there are at least four lesser clusters of TFIIB cross-links upstream from the predominant TFIIB peak, raising the possibility of less abundant PICs at alternative sites. Consistent with this, TBP shows a broad peak of



FIG 5 TBP DNA binding surface mutants function in vitro at the TATA-less RPS5 promoter. In vitro transcription using TBP mutants is shown. Transcription


DISCUSSION

FIG 6 In vitro transcription from RPS5 is TFIID dependent and SAGA independent. (A) In vitro transcription from the RPS5 promoter using either WT or taf11 ts WCE as indicated. Reaction mixtures contained either 180 ␮g (lane 1) or 240 ␮g (lanes 2 to 11) WCE, and all reaction mixtures contained Gal4VP16. TFIID (0.16 to 0.64 ␮g), SAGA (0.13 to 0.52 ␮g), or recombinant TBP (rTBP) (24 to 72 ng) was added as indicated. (B) In vitro RPS5 transcription as in panel A, except either WT extract or extract made from a ⌬spt7 strain was used. Gal4-VP16 and 250 ␮g WCE were added to all reaction mixtures. TAPtagged purified SAGA was added where indicated. Transcription in panels A and B was visualized by RT with 32P-labeled lacI primer.

cross-linking at RPS5, encompassing the sites of FP1, FP2, and FP3 with the highest levels of cross-linking observed at FP2 and FP3. Unfortunately, attempts to obtain a footprint of TFIID over the RPS5 promoter were unsuccessful, even though the preparation of TFIID used was active in transcription at RPS5, possibly because the concentration of our purified TFIID was not high enough. Our TBPc footprints at RPS5 seem identical to those of a published TFIID footprinting study (42), which suggests that either the previous work mapped only TBP binding rather than TFIID or that the TFIID Taf subunits do not contribute to the footprint pattern at RPS5. Although we found that disruption of the TBP DNA binding surface had little effect on RPS5 transcription, the discovery of TBP binding sites on the RPS5 promoter prompted the question of whether they make a functional contribution to transcription. To address this, we created a series of mutant RPS5 promoters designed to disrupt the two most prominent TBP binding sites by deletion of one or both sites (Fig. 9A). As expected from the minimal effects of TBP DNA binding surface mutations on RPS5 transcription, plasmids containing deletions of the individual or combined TBP footprints had wild-type levels of activity in vitro


The two general types of Pol II promoters, TATA-containing and TATA-less promoters, both require TBP, but there are conflicting data on whether TBP directly contacts DNA in a sequence-specific fashion at TATA-less promoters. On one hand, TATA-related sequences with up to two or three mismatches to the TATA consensus are tightly linked to the positions of TBP/TFIIB cross-linking (35, 47). However, the coactivator TFIID is typically required for transcription of TATA-less genes, and one or more alternative promoter elements, such as Inr, DPE, and MTE, are required for metazoan transcription in the absence of a consensus TATA. In addition, a human TBP mutant that is defective in TATA binding was found to be specifically defective for in vitro transcription only at TATA-containing promoters (57). Coactivator specificity is usually determined by the core promoter sequence. However, the determinants that make a yeast promoter TFIID dependent are unknown, in part because specific TFIID binding motifs analogous to the metazoan Inr, DPE, and MTE have not been identified. The yeast GA element, a sequence of unknown function that is found in many TATA-less promoters (10), is not present in the RPS5 promoter, and there is no evidence yet that it is involved in TFIID binding. The lack of identified TFIID binding motifs in yeast has left open the possibility that specific TBP binding at TATA-less promoters plays a role in TFIID targeting. Here, we have investigated the role of sequence-specific TBP binding in transcription of TATA-less genes, focusing mainly on the well-studied RPS5 ribosomal protein gene. We found that many mutations in conserved TBP residues involved in DNA binding at TATA had little effect on transcription of two TATA-less genes while showing significant transcription defects at two TATA-containing genes. We replicated these findings in an in vitro system, showing that the selective defect in TATA-containing promoter transcription was not due to an indirect effect or specific to the transcription activator used. Our results also agree with the in vitro findings in the human system, despite there being no obvious TFIID recognition elements in yeast promoters. Although we surprisingly found that TBP bound to several TATA-less genes in vitro, our studies suggest that this binding is not relevant for transcription of these genes. First and most importantly, the TBP mutants tested are defective for binding to both the TATA-containing HIS4 and the TATA-less RPS5 promoters but still promote transcription from two TATA-less genes. Second, deletion of the two principal TBP binding sites within RPS5 has no effect on in vitro transcription. Our results are strikingly consistent with the results of two other studies examining the role of TBP in activated transcription

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(Fig. 9B and C). In addition, the positions of the TSSs were not altered in any of the mutant promoters (Fig. 9B). These findings are consistent with in vivo results, which showed that disruption of these regions had no effect on in vivo RPS5 transcription (41). Surprisingly, we found that TBP bound to alternative AT-rich segments of the RPS5 promoter containing the double deletion of TBP binding sites (Fig. 9D and E). Overall, our findings suggest that even though TBP can bind to specific sites at the RPS5 promoter, the mechanism of how it initiates PIC formation differs from the one described for TATA-containing promoters, as neither the sequence-specific TBP DNA binding surface nor the two most prominent DNA sites normally bound by TBP in vitro play a significant role in RPS5 transcription.

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(2 ng) was incubated with labeled HIS4 promoter and the indicated molar excess of unlabeled DNA. Binding is normalized to the level observed in the absence of competitor. The unlabeled competitor probes used are HIS4 (identical to labeled probe), HIS4mut (HIS4 promoter with mutant TATA box), and RPS5 (RPS5 promoter). (B) TBP-HIS4 EMSA as in panel A with the indicated unlabeled competitor promoter fragments. (C) EMSA with radiolabeled RPS5 and HIS4 promoters using WT TBP or TBP core (TBPc). Purified recombinant wild-type TBP (2 ng) or TBPc (2 ng) was used and recombinant TFIIA (IIA) (2 ng) was added as indicated. (D) Electrophoretic mobility shift assay with radiolabeled double-stranded RPS5 promoter DNA probe. EMSA as in panel C but with either WT TBPc or the DNA binding defective TBPc derivatives as indicated.

(52, 59). In these studies, a number of TBP mutations were isolated; these mutants showed defects in activated transcription from several natural genes and reporter constructs. These TBP mutants were defective for in vitro TATA DNA binding, but they supported yeast cell growth and showed little or no transcription defects at constitutively expressed genes. In retrospect, the inducible genes assayed were all TATA-containing genes, while the unaffected genes contained TATA-less promoters and/or were TFIID-dependent genes (9, 20). Combining our results with the findings of these earlier studies strongly suggests that the sequence-specific mode of TBP-DNA interaction is not required for transcription at TATA-less TFIID-dependent promoters—the majority of cellular promoters. The DNA binding surface of TBP also interacts with Mot1, the Taf1 TAND1 domain, and another molecule of TBP in the TBP dimer. However, the phenotypes of the TBP mutants and the wide range of TBP mutations that show differential transcription of

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TATA and TATA-less genes strongly suggest that the effects we observe are mediated by defective TBP-DNA interactions. First, mutations in the Mot1 latch domain that interact with the TBP DNA binding surface show defects in TBP-DNA displacement (53), and Mot1 defects are known to increase TATA-dependent expression at the expense of TATA-less expression (71), opposite to the results observed here. Second, mutation of the Taf1 TAND1 domain results in very modest changes in global gene expression (55), in contrast to our findings at two TATA-containing genes. Third, disruption of TBP dimerization can result in increased basal transcription with only mild defects in induced transcription levels (56), again in contrast to what we observe here. Finally, four of the residues mutated here (R196, V203, L205, and F207) are not known to be involved in either Mot1 or TAND1 binding, yet they show differential transcription of TATA and TATA-less genes. It is important to note that in vivo, TBP does not efficiently bind either TATA or TATA-less promoters without assistance



FIG 7 TBP binds to several TATA-less promoters. (A) EMSA with radiolabeled HIS4 promoter probe and unlabeled competitor DNA probes. Wild-type TBPc


from a coactivator. For example, many uninduced yeast TATAcontaining promoters have low TBP occupancy, and efficient transcription requires binding of an activator and the function of the coactivator SAGA (26, 72–74). Likewise, recruitment of TBP to TATA-less promoters requires the coactivator TFIID (25), which is recruited to many housekeeping genes by gene-specific regulatory factors. For example, it was recently shown that TFIID function at yeast ribosomal protein genes involves an interplay between the activator Rap1, TFIIA, and the TFIID subunit Taf4 (75). The apparent difference between these two promoter types is that the specific TBP-TATA interface is critical for expression at the TATA-containing genes, while mutations that disrupt TBPTATA binding have little effect on the TATA-less genes. The simplest model to explain the results in the yeast system is similar to that proposed for human transcription in which TFIID can specifically bind promoters via alternative promoter elements without the need for specific TBP-DNA contact (57). It has been found that insertion of a TATA motif into a TFIID-dependent promoter gives a synergistic increase in transcription output (8, 57, 76). Therefore, TFIID may have two binding modes: (i) a binding mode where TBP and the Tafs make sequence-specific pro-


moter contacts and (ii) a binding mode at TATA-less promoters where only the Tafs make sequence-specific interactions with the Inr and other alternative elements. However, this model leaves open the question of whether TBP is required to make non-sequence-specific contacts at TATA-less promoters. If there are nonspecific DNA interactions, they must occur through an interface much different than TBP-DNA binding at TATA-containing promoters which involves opening of the minor groove and DNA kinking at either end of the TATA. This is because mutations in TBP residues F116 and F207, critical for DNA kinking and the sharp bend observed in the TBPTATA structure (11, 12), can be mutated with little effect on transcription from RPL5 and RPS5. Relevant to this question are two recent studies that examined the structure of TFIID bound to promoters using cryo-electron microscopy (cryo-EM) (31, 77). Although the resolution was too low to directly observe TBP-DNA contacts at TATA-containing promoters, the path of DNA and mapped position of TBP suggest that TBP and TATA are in close proximity. Interestingly, the DNA path and overall structure of the human TFIID-promoter complex did not appear altered when bound to a promoter with a

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FIG 8 TBP binds to the RPS5 promoter at two distinct sites. DNase I footprint of RPS5 and HIS4 promoters. TBPc (15 to 37.5 ng for RPS5 and 62.5 ng for HIS4) and TFIIA (10 ng) were added as indicated. The locations of the TBP footprints with respect to the RPS5 TSS are shown at the bottom of the figure. The major in vivo TSS is underlined.

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mutated TATA (31), raising the possibility of important non-sequence-specific TBP-DNA contacts at TATA-less promoters. Since human TFIID undergoes a large structural change upon binding to DNA in the active form (31), non-sequence-specific TBP-DNA interactions may play a role in stabilizing this conformation and explain why TBP is required for transcription at TATA-less genes. Finally, what is the role of the TATA-like sequences found near the sites of TBP/TFIIB cross-linking? Structural and bio-

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chemical studies have led to models for the structure of the Pol II PIC at TATA-containing promoters (1). A key feature of the TATA PIC structure is the ⬃90° DNA bend centered on the TATA. This allows the TFIIB core domain to bind the bent DNA on either side of TBP and subsequently interact with the surface of Pol II to set the initial path of promoter DNA across the Pol II cleft. Although the structure of a TFIID-containing PIC on TATA-less DNA has not been modeled, it seems likely that the interaction of TFIIB with Pol II will be conserved as



FIG 9 The two principal TBP binding sites at the RPS5 promoter are dispensable for in vitro transcription. (A) Mutant RPS5 promoter constructs tested for transcription in vitro. The wild-type sequence of the TBP binding sites is shown below the RPS5 schematic. Constructs in which 8 bp of either or both TBP binding sites are deleted are named ⌬FP1, ⌬FP2, and ⌬FP1⌬FP2. Deleted nucleotides are indicated by dashes. (B) Representative in vitro transcription assay comparing wild-type RPS5 and mutant promoter activity. Each construct was tested in duplicate. The results are visualized by primer extension with radiolabeled lacI primer and resolved on a sequencing gel. The positions of the two major transcription start sites used in quantification are marked. (C) Quantification of in vitro transcription activity of RPS5 mutant promoter constructs from panel B. Transcripts from the major upstream (TSS1) and downstream (TSS2) start site groups were measured and normalized to the levels of the wild-type RPS5 promoter. Error bars represent standard deviations. (D) DNase I footprint of RPS5 ⌬FP1⌬FP2 mutant promoter in the presence of recombinant TBPc and TFIIA. The footprint regions are marked by brackets. The TBP binding sites on RPS5 ⌬FP1⌬FP2 are named FP1* (upstream) and FP2* (downstream). (E) Locations of the TBP footprints on the RPS5 ⌬FP1⌬FP2 promoter. The locations of the two 8-bp deletions shown in panel A are indicated.


12. 13. 14. 15.

16. 17.

ACKNOWLEDGMENTS We thank B. Knutson and J. Fishburn for help with RNA analysis, protein expression, protein purification, and in vitro transcription assays, S. Buratowski for the TAF11 ts strain, P. A. Weil’s laboratory for advice on preparing whole-cell extracts, F. Pugh and H. S. Rhee for sharing their ChIP-exo data and for discussions, J. Geiger for discussions on TBP binding mechanisms, and B. Knutson and S. Grünberg for comments on the manuscript. This work was supported by grant 2RO1GM053451 from the NIH to S.H.

18.

19.

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TATA-dependent and TATA-independent transcription at the HIS4 gene of yeast.

SHI, a new yeast gene affecting the spacing between TATA and transcription initiation sites.

Yeast and human TATA-binding proteins have nearly identical DNA sequence requirements for transcription in vitro.

Molecular Mechanism of Mot1, a TATA-binding Protein (TBP)-DNA Dissociating Enzyme.

Mot1 redistributes TBP from TATA-containing to TATA-less promoters.

A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1.

Interaction between TATA-Binding Protein (TBP) and Multiprotein Bridging Factor-1 (MBF1) from the Filamentous Insect Pathogenic Fungus Beauveria bassiana.

The conformational state of the nucleosome entry-exit site modulates TATA box-specific TBP binding.

Cip1) gene depends on transcription factor IIA (TFIIA) in addition to TFIIA-reactive TBP-like protein.

Direct interaction between adenovirus E1A protein and the TATA box binding transcription factor IID.

Functional binding of the "TATA" box binding component of transcription factor TFIID to the -30 region of TATA-less promoters.

Yeast and human TFIID with altered DNA-binding specificity for TATA elements.

Transcription factor TFIID induces DNA bending upon binding to the TATA element.

Regulation of anti-sense transcription by Mot1p and NC2 via removal of TATA-binding protein (TBP) from the 3'-end of genes.

Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription.

Transcription. Riding high on the TATA box.

A TBP complex essential for transcription from TATA-less but not TATA-containing RNA polymerase III promoters is part of the TFIIIB fraction.

Real-Time Interaction between TBP and the TATA Box of the Human Triosephosphate Isomerase Gene Promoter in the Norm and Pathology.

Regulation of delayed-early gene transcription by dual TATA boxes.

Isolation of a yeast gene encoding a protein homologous to the human Tat-binding protein TBP-1.

An element of symmetry in yeast TATA-box binding protein transcription factor IID. Consequence of an ancestral duplication?

Transcription factors: specific DNA binding and specific gene regulation.

A suppressor of TBP mutations encodes an RNA polymerase III transcription factor with homology to TFIIB.

TBP-like protein (TLP) interferes with Taspase1-mediated processing of TFIIA and represses TATA box gene expression.