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Originally published In Press as doi:10.1074/jbc.M508253200 on November 1, 2005

J. Biol. Chem., Vol. 281, Issue 1, 80-89, January 6, 2006
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Mediator as a General Transcription Factor*{boxs}

Yuichiro Takagi and Roger D. Kornberg1

From the Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5400

Received for publication, July 28, 2005 , and in revised form, October 31, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Others have shown that yeast strains bearing a ts mutation in the Srb4 subunit of Mediator cease transcription of all mRNA at the restrictive temperature, in a manner virtually indistinguishable from a strain bearing a ts mutation in the largest subunit of RNA polymerase II. We find that srb4ts Mediator is defective for the stimulation of basal RNA polymerase II transcription at the restrictive temperature in vitro. Taken together, these findings lead to the suggestion that Mediator is required for basal RNA polymerase II transcription in vivo. On this basis, Mediator is identified as a general transcription factor, comparable in importance to RNA polymerase II and other general factors for the initiation of transcription. The possibility that Mediator serves as an anti-inhibitor, opposing the effects of global negative regulators, is largely excluded.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mediator was discovered in 1990 in a yeast transcription system (1) and shown to support transcriptional activation (2). Purification of Mediator to homogeneity (3) led to the finding of two further activities, stimulation of basal transcription and stimulation of TFIIH protein kinase. The occurrence in Mediator of certain subunits, implicated by genetic screens in the negative regulation of transcription, revealed yet another Mediator activity, its function as a co-repressor (4–6).

Co-activators were reported in a human transcription system in 1991 but were believed at the time to be unrelated to yeast Mediator (7). Attention focused instead on TAFs as responsible for the transmission of regulatory information in metazoan systems (8). It was only in 1998 that protein complexes in mammalian cells were identified as counterparts of yeast Mediator and shown to support transcriptional activation (9–13). Human Mediator has since been reported to stimulate basal transcription as well (14, 15).

Studies in the human system provided evidence for direct activator-Mediator interaction. Thyroid hormone receptor (16), SREBP (12), adenoviral E1A (17), and other transcriptional regulatory proteins were isolated from human cells as tight complexes with Mediator. Activator-Mediator interaction has been reported in the yeast system (18–20), but tight complexes have not yet been demonstrated in yeast cell extracts or in derived fractions.

Mediator also interacts directly with RNA polymerase II (pol II).2 A Mediator-pol II complex was initially isolated from yeast (3, 21) and has been verified by both structural (22, 23) and functional analyses. A chain of communication from activator to Mediator to pol II is therefore believed to underlie transcriptional activation (24–29).

Mediator-pol II interaction may also underlie the stimulation of basal transcription. Genetic studies, however, have lent credence to an alternative mechanism. Temperature-sensitive mutants in two Mediator subunits, Srb4 and Srb6, exhibit a remarkable phenotype at a restrictive temperature, the rapid cessation of transcription of all genes tested (30). This finding has been extended by microarray analysis to 5361 yeast genes, all but two of which showed as great an effect of the srb4ts mutant on transcription as that of a mutation in pol II itself (31). A screen for suppressors of the srb4ts mutation identified subunits of the previously described Not and NC2 protein complexes (32, 33). These complexes are believed to be inhibitors of transcription, raising the possibility that Mediator serves as an anti-inhibitor. Mediator might stimulate basal transcription by relieving repression due to global negative regulatory factors.

We report here on a study of the srb4ts mutant Mediator in vitro. The results are informative about the mutant phenotype and the role of Mediator in vivo. They provide a test of the anti-inhibitor idea and illuminate the Mediator mechanism, especially in regard to the stimulation of basal transcription.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reversion of srb4ts Mutations and Phenotypic Analysis—The yeast shuttle vector pCT181, carrying the srb4–138 mutant allele, was recovered from yeast strain Z628 (30). The shuttle vector pCT127, carrying the wild type SRB4 gene, was recovered from the isogenic yeast strain Z579. The mutations in pCT181 were reverted one at the time by QuikChange (Qiagen), and the sequences were verified by DNA sequencing. Then the shuttle vectors were introduced into yeast strain Z572 by plasmid shuffling. For controls, the wild type vector, pCT127, and the mutant, pCT181 were also introduced into yeast strain Z572. The isolated yeast strains were grown in YPD (2% (w/v) Bacto Peptone, 1% (w/v) yeast extract, 2% (w/v) glucose) overnight. The cells (3 µlof culture diluted to an A600 of 1) were streaked on YPD plates and grown at 30 or 37 °C.

Expression Vectors—The open reading frames of yeast SRB6, ROX3, and MED3 genes were amplified by PCR and cloned between BamHI and XhoI sites, EcoRI and XhoI sites, and SmaI and XhoI sites of pGEX6P-1 (Amersham Bioscience), respectively, yielding the expression vectors, pTK006(GST-Srb6), pTK009(GST-Rox3), and pTK067 (GST-Med3). The open reading frames of yeast SRB2, SRB5, and MED11 genes were amplified by PCR and cloned between NdeI and BamHI sites of pET16b (Novagen), yielding the expression vectors pTK021(10His-Srb2), pTK029(10His-Srb5), and pTK038(10His-Med11), respectively.

Recombinant Mediator Subunits—Expression and purification of the GST fusion proteins GST-Srb6, GST-Rox3, and GST-Med3 were performed as described (34) with one minor modification; instead of eluting with glutathione-containing buffer, the recombinant Srb6 was released from the column by cleavage of GST-Srb6 protein with PreScission protease according to the manufacturer's instructions (Amersham Bioscience). 10His-Srb2, 10His-Srb5, and 10His-Med11 were expressed and cell lysates were made by essentially the same procedure except the lysis buffer was phosphate-buffered saline, 10 mM imidazole (pH 8.0), 300 mM NaCl, 10% glycerol, 0.01% Nonidet P-40, 5 mM {beta}-mercaptoethanol, protease inhibitor mix. The lysates were cleared by centrifugation at 100,000 x g for 60 min and supernatants were loaded on a 1 ml column of nickel-nitrilotriacetic acid resin (Qiagen) equilibrated with lysis buffer. For 10His-Srb2 and 10His-Srb5 proteins, after washing with 50 ml of lysis buffer containing 20 mM Imidazole (pH 8.0), proteins were eluted with lysis buffer containing 300 mM imidazole (pH, 8.0). 10His-Med11 was eluted with lysis buffer containing 8 M urea and 300 mM imidazole (pH 8.0).

Antibody Production and Immunoblot Analysis—About 1 mg of each of the recombinant proteins Srb6, GST-Rox3, 10His-Srb2, and 10His-Srb5 inoculated in rabbits (Covance). GST-Med3 and 10His-Med11 were fractionated by SDS-PAGE and revealed by staining with Coomassie Brilliant Blue R-250. The GST-Med3 and 10His-Med11 protein bands were excised and inoculated in rabbits (Covance). Antibodies against Srb4 and Rgr1 proteins were made by inoculating rabbits with the peptides, DNDKNLKFLKNKDSLV (Srb4 amino acids 72–87) and CMEIHNILKVDSNSSSS (cysteine plus Rgr1 amino acids 1067–1081), conjugated with keyhole limpet hemocyanin (Covance).

Anti-Sin4 antibody was gift from D. Stillman (University of Utah). Anti-Med1 was a gift from S. Bjorklund (Umea University, Umea, Sweden). Anti-Med2, Med4/5, Med6, Med7 and Med8 antibodies were as described (Myers et al. (6)). Anti-Med9 and Med10 were gifts from C. Gustafsson (Karolinska Institute, Huddinge, Sweden). Anti-Not1, Not3, Not5 and Caf40 were gifts from C. Denis (Universtiy of New Hampshire). Anti-Mot1 was a gift from D. Auble (University of Virginia). Anti-NC2 antibodies were a gift from M. Collart (University of Geneva, Geneva, Switzerland). Immunoblot analysis was performed essentially as described (34).

Construction of Yeast HA-tagging Vector—The HA-tagging vector pYT006 was created by modifying the vector pU6H3HA (35) as follows. The six-histidine tag was disabled by mutating the first four histine residues to glycines, yielding pYT005, followed by introducing the sequence of the PreScission protease site (LEVLFQGP) before the three copies of the HA epitope by QuikChange (Strategene), yielding pYT006. The BamHI fragment from the vector pDp-U URA (36), containing the URA3 gene, was blunted and subcloned into the blunted ApaI and Hin-dIII sites of pBS1479 (37), yielding the URA3 marker-containing vector pYT0(A). The tabacco etch potyvirus protease cleavage site was replaced by a PreScission protease site (LEVLFQGP) by subcloning the corresponding DNA sequences into NheI and SacI sites of pYT0(A), yielding the yeast tagging vector pYT001(A).

Construction of Tagged Yeast Strains—A 10-histidine tag was introduced at the N termini of wild type and mutant srb4 genes in pCT127 and pCT181 by QuikChange (Strategene), yielding pCT127(10His-Srb4) and pCT181(10His-srb4ts) respectively. Three copies of the HA epitope were introduced at the C terminus of Med8 in both wild type and srb4 mutant yeast strains by PCR from pYT006 with primer sets targeting the Med8 genomic locus as described (35). The PCR products were used to transform yeast strain Z572 (MATa leu2–3, 112 ura3–52 [CEN, URA3, SRB4], Med8:Med8-PreScission-3xHA-Kan), yielding yeast strain YT108. Finally, pCT127(10His-Srb4) and pCT181(10His-srb4ts) were transformed into YT108 by plasmid shuffling, yielding the yeast strains YT110(10His-Srb4, Med8-PreSci-3xHA) and YT111(10His-srb4ts, Med8-PreSci-3xHA), respectively.

Following the published protocol (37), PCR was performed with pYT1(A) as template and with primer sets targeting Not1 genomic locus. The PCR products were used to transform yeast strain Z579 [MATa leu2–3, 112 ura3–52 , Not1:Not1-mTAP-URA3], yielding the yeast strain YT022(NotI-mTAP).

Purification of Wild Type and srb4ts Mediators—Yeast strain YT110(wt:Med8-PreSci-3xHA) or YT111(srb4ts:Med8-PreSci-3xHA) was grown in 20 liters of 2x YPD (4% (w/v) Bacto Peptone, 2% (w/v) yeast extract, 4% (w/v) glucose) to an A600 value of 8–9. Frozen cells (1.2 kg) were broken in liquid nitrogen as described (34) in a 2-liter Waring blender at high speed for 10 min with constant addition of liquid nitrogen. A portion of broken cells (350 g) were thawed at 4 °C and 500 ml of 0.27 M Tris acetate (pH 7.6), 0.95 M potassium acetate, 1.8 mM EDTA, 18% glycerol, 10 mM {beta}-mercaptoethanol, protease inhibitor mix was added. The mixture was stirred at 4 °C for 30 min and clarified by centrifugation in a Beckman JA14 rotor at 13,000 rpm for 20 min and then in a Beckman Ti45 rotor at 42,000 rpm for 90 min. The supernatant was dialyzed against the buffer A (50 mM Hepes-KOH (pH 7.6), 10% glycerol, 5 mM {beta}-mercaptoethanol) for 3 h, adjusted to a conductivity of buffer A containing 100 mM potassium acetate ("buffer A+100"), with the buffer A, and applied to a 500 ml of BioRex 70. Step elution was performed with buffer A+300, +600, and +1200 as described (3). The buffer A+600 eluates from three portions of broken cells processed in this way were combined and dialyzed against the buffer B (50 mM Tris acetate (pH 7.6), 0.1 mM EDTA, 0.01% Nonidet P-40, 10% glycerol, 5 mM {beta}-mercaptoethanol) for 2 h, adjusted to the conductivity of the buffer B containing 100 mM potassium acetate ("buffer B+100") with the buffer B, and then applied to a 100-ml DEAE-Sephacel column pre-equilibrated with buffer B+100. The column was washed with 1 column volume of buffer B+100 and eluted with a linear gradient from 100 mM to 550 mM potassium acetate over 10 column volumes. The peak free Mediator fractions eluted at ~400 mM potassium acetate.

DEAE-Sephacel fractions containing free Mediator (~160 ml) were concentrated by centrifugation with a PL-10 (Amicon) to 10 ml, 7.55 mg/ml (wild type Mediator) or 7 ml, 12.8 mg/ml (srb4ts Mediator). (It proved to be essential to concentrate the DEAE fractions before loading onto an HA affinity column.) Half of the concentrated DEAE fraction was loaded on a 0.8-ml anti-HA antibody column (Sigma) pre-equilibrated with buffer A+300 containing 0.01% Nonidet P-40 and protease inhibitors. After 2 h at 4 °C, the column was washed with 80 ml of buffer A+300 containing 0.01% Nonidet P-40. (To maintain the integrity of srb4ts Mediator, it was essential to use potassium acetate at a concentration no higher than 300 mM. Use of ammonium sulfate or a higher ionic strength resulted in a loss of subunits.) The column was equilibrated with 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 0.01% Nonidet P-40, 5 mM {beta}-mercaptoethanol, and eluted by incubation for 1.5 h at room temperature in the same buffer containing PreScission protease (8 units/ml) and 2x HA peptide CPDYAGYPYDVPDYAGYPYDV (0.2 mg/ml). The combination of 2x HA peptide and protease digestion was necessary to obtain an elution efficiency of greater than 90%. Use of only one eluant resulted not only in a poor yield but also in loss of subunits from srb4ts Mediator. The elute was dialyzed against 50 mM Hepes-KOH (pH 7.8), 150 mM KOAc, 20% glycerol, 5 mM dithiothreitol for 1 h, and 5–10 µl was subjected to immunoblot analysis.

Transcription and C-terminal Domain (CTD) Phosphorylation Assays—Whole cell extracts of wild type (Z579) and srb4–138 (Z628) strains (30) were prepared and used for transcription as described (38). Transcription reconstituted with purified proteins was performed as described (3) with the following modifications. Nucleotide mix (ATP, CTP, UTP) and magnesium acetate were added only following a preincubation period. The final concentration of cold UTP was 10 µM instead of 25 µM. Where Sarkosyl was added, the final concentration was 0.2%. For quantitation of transcripts on an absolute rather than relative basis, 1 nCi of [{alpha}-32P]UTP was applied to the gel, 5 min before the end of the run. Quantitation was performed with a PhosphorImager and ImageQuant software. The CTD phosphorylation assay was performed as described (3).



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  		FIGURE 1.
An srb4ts mutant yeast extract is temperature-sensitive for basal pol II transcription in vitro. Wild type and srb4ts mutant yeast extracts (60, 120, and 240 µg in lanes 1–3 and repeated every three lanes) were prepared and assayed with pJJ470 template (Fig. 2A) as described (see"Materials and Methods") at 24 °C (lanes 1–6) or 30°C (lanes 7–12). An autoradiograph of the gel is shown. Lane M, 32P-labeled {phi}X/Hinf III DNA markers with sizes (bp) indicated on the left; wt, wild type extract; srb4ts, mutant extract. Radioactivity in transcripts, determined by PhosphorImager analysis, is shown below.

 



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  		FIGURE 2.
Wild type (wt) Mediator restores transcription activity to srb4ts mutant yeast extract at the restrictive temperature. Transcription was performed as in Fig. 1, lane 9 (lane 1), or Fig. 1, lane 12 (lanes 2–24) with the addition of wild type Mediator (0.1, 0.2, and 0.4 pmol in lanes 3–5, respectively), TBP (2.2 and 4.3 pmol in lanes 6 and 7, respectively), TFIIB (1 and 2 pmol in lanes 8 and 9, respectively), TFIIF (0.6 and 1.3 pmol in lanes 10 and 11, respectively), TFIIE (0.5 and 1.1 pmol in lanes 12 and 13, respectively), TFIIH (2.2 and 4.3 pmol in lanes 14 and 15, respectively), pol II (0.4 and 0.7 pmol in lanes 16 and 17, respectively) (lanes 6–17), or combinations of additional proteins as indicated above lanes 18–24 (TBP, 2.2 pmol; TFIIB, 1 pmol; TFIIF, 0.6 pmol; TFIIE, 0.5 pmol; TFIIH, 2.2 pmol; pol II, 0.4 pmol).

 
Purification of NOT Complex—The preparation from the Not1-mTAP tagged yeast strain YT022 was performed as described (34) with the following modifications: 1 M potassium acetate was used instead of ammonium sulfate in the lysis buffer. After the centrifugation at 100,000 x g, for 90 min, the extract was dialyzed against 50 mM Hepes-KOH (pH 7.8), 150 mM potassium acetate, 20% glycerol, 5 mM {beta}-mercaptoethanol overnight, centrifuged at 100,000 x g for 30 min, and loaded on a 1-ml IgG-agarose column (Sigma) equilibrated in buffer A containing 600 mM potassium acetate at 4 °C. After loading, the column was washed with 100 ml of buffer A+600 containing 0.01% Nonidet P-40 and protease inhibitors and 100 ml of buffer A+150 mM containing 0.01% Nonidet P-40. The column was equilibrated with 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 0.01% Nonidet P-40, 5 mM {beta}-mercaptoethanol, and eluted by incubation overnight in the same buffer containing PreScission protease (8 units/ml) at 4 °C. IgG elutes were further purified on a 0.5-ml calmodulin column as described (37) except that both calmodulin binding buffer and elution buffer contained 0.01% Nonidet P-40 and 5% glycerol. The calmodulin column elute was dialyzed as described above.



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  		FIGURE 3.
Mediator is required for the initiation of transcription. A, design of experiment. Transcription was performed with srb4ts mutant yeast extract as in Fig. 1, lane 9, with the addition of purified wild type (wt) Mediator (100 ng and 0.1 pmol) at the times indicated and with the addition of Sarkosyl 2 min after nucleoside triphosphates to prevent reinitiation. Transcripts were quantitated and plotted as a function of the time of wild type Mediator addition in B. WCE, whole cell extract.

 
Expression and Purification of Yeast NC2—Bur6–6His and 6His-Ydr1 were overexpressed as described above. The expression vectors for NC2, pGP288, and pGP415 were a gift from G. Prelich (39). After centrifugation, the pellet containing the inclusion bodies was resuspended in 20 ml of denaturation buffer (phosphate-buffered saline, 6 M guanidine-HCl, 500 mM NaCl, 10 mM imidazole), clarified by centrifugation, and loaded on a 2-ml column of nickel-nitrilotriacetic acid pre-equilibrated with denaturation buffer. The column was washed with 50 ml of denaturation buffer and 50 ml of denaturation buffer containing 20 mM imidazole and was eluted with denaturation buffer containing 300 mM imidazole.

For reconstitution of NC2, equal amounts of both nickel column elutes were mixed, diluted 1:5 with renaturation buffer (50 mM Hepes-KOH (pH 7.8), 0.1 mM EDTA, 150 mM KCl, 50 µM zinc acetate, 2 mM dithiothreitol, 10%glycerol), placed on ice for 90 min, dialyzed against 50 mM Hepes-KOH (pH 7.6), 150 mM potassium acetate, 50 µM zinc acetate, 10% glycerol, 2 mM dithiothreitol, and loaded on a 1-ml Hitrap Q column pre-equilibrated with buffer A+100. The recombinant NC2 was eluted by a linear gradient of 100 mM to 1 M potassium acetate. The peak fraction was dialyzed as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transcription by srb4ts Mutant Cell Extract Correlates with the Mutant Phenotype, Implying a Role for Mediator in Basal Transcription in Vivo—Whole cell extracts were prepared from wild type and srb4ts yeast strains as described. The extracts were nearly identical in total protein concentration, in concentrations of general transcription factors and Mediator (including Srb4 protein; all concentrations measured by immunoblot analysis), and in nonspecific pol II activity. Assayed at a permissive temperature of 24 °C, the extracts supported comparable levels of promoter-dependent transcription from a commonly used template, the CYC1 promoter fused to a G-less cassette (Fig. 1). At a restrictive temperature of 30 °C, transcription by the srb4ts extract was 23 times less than that by the wild type extract. The inhibition of transcription in the mutant extract was reversible; upon warming to 30 °C or even 37 °C and then cooling to 24 °C, transcription was restored to the wild type level (data not shown). Inhibition was therefore not a consequence of degradation or the like (important to rule out, as a previous observation regarding transcription of an immobilized template by srb4ts extract (40) raised the possibility of loss of Srb4 protein in the mutant in vivo; this observation may be attributed to washing of the immobilized preinitiation complex prior to transcription, resulting in loss of Srb4 protein, due to instability of the mutant Mediator in vitro described below).



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  		FIGURE 4.
Mediator requirement is satisfied (Mediator can be inactivated) 10 min prior to the initiation of transcription. A, design of experiment. Transcription was performed with srb4ts mutant yeast extract as in Fig. 1, lane 6, with transfer of the reaction from 24 to 30 °C at the times indicated. Transcripts were quantitated and plotted as a function of the time of temperature increase in B. Horizontal lines across the plot indicate values expected for single (Fig. 3, Mediator added before NTPs) and multiple (Fig. 1, lane 9) rounds of transcription. WCE, whole cell extract.

 
Similar results were obtained with a variety of transcription templates (supplemental figure). Transcription by the mutant extract was temperature-sensitive, regardless of the promoter (CYC1 or adenoviral major late) or the UAS (Gal4-binding, Gcn4-binding, or none) used. As no activator protein was added, and no UAS was required, the stimulation of basal transcription by Mediator was evidently affected. From the correlation of this biochemical behavior with the mutant phenotype, we conclude that Mediator performs a fundamental role in transcription in vivo. It is important not only for regulated transcription but, apparently, for any transcription at all.

Mediator Performs a Unique Role in the Initiation of Transcription—Addition of purified wild type Mediator to srb4ts extract restored transcription activity at 30 °C (Fig. 2). In contrast, addition of general transcription factors and pol II, individually or in various combinations, at levels greater than those in the extract, was without effect. We conclude the loss of activity in the srb4ts extract at 30 °C was directly attributable to the loss of Mediator function and not due indirectly to an influence of the mutant Mediator upon the expression of another factor. We further conclude the role of Mediator in basal transcription is distinct from the roles of the other transcription proteins.

We could exploit the restoration of activity by wild type Mediator to determine when Mediator acts on the transcription pathway. Mediator was added to srb4ts extract at 30 °C at various times before or after the addition of NTPs. Sarkosyl was added 2 min after NTPs to prevent further initiation and allow only RNA chain elongation (Fig. 3). The results were clear-cut; addition of Mediator 5 min or longer before NTPs gave full transcription activity, whereas addition at the same time as NTPs or any time after gave no transcription at all. We conclude that Mediator functions in the initiation of transcription.

In other experiments, the temperature was raised from 24 to 30 °C at various times before or after transcription the addition of NTPs to srb4ts extract (Fig. 4). Temperature increase 20 min or more before NTP addition resulted in no transcription at all. A temperature increase between 10 min before and 10 min after NTP addition yielded a level of transcription comparable with that following Sarkosyl addition at 2 min of reaction in wild type extract, indicative of a single round of initiation and transcript elongation (data not shown). Temperature increase at later times permitted multiple rounds of transcription. This provided further evidence for a role of Mediator in initiation, and demonstrated a lack of involvement of Mediator in transcript elongation. It furthermore demonstrated a requirement for Mediator for multiple rounds of transcription.



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  		FIGURE 5.
Six mutations are required for the temperature-sensitive phenotype of the srb4ts yeast strain. A, schematic of SRB4 gene showing locations of mutations (residue numbers above) in the srb4ts allele used in this work, with amino acid changes for reversion to wild type indicated below. B, wild type, srb4ts, and six single amino acid revertants were grown in YPD medium, spotted onto YPD plates, and grown either at 30 or 37 °C as indicated. C, temperature sensitivity of single amino acid revertants analyzed as in B. D, temperature sensitivity of double and triple amino acid revertants analyzed as in B. WT, wild type.

 
Genetic and Biochemical Characterization of srb4ts Mediator Supports a Direct, Positive Role in Transcription, as Opposed to Inhibition at Restrictive Temperatures and Anti-inhibition at Permissive Temperatures—Sequencing the srb4ts allele revealed eight amino acid differences from wild type (Fig. 5A). To determine which difference(s) was responsible for the temperature-sensitive phenotype, the mutant residue was reverted to wild type at each of the eight positions individually, in pairs, and in a triple revertant. Three of the individual revertants lost temperature sensitivity (Fig. 5, B and C), as did the triple revertant (Fig. 5D), identifying six amino acid changes (L21S, M313I, S226P, E460G, E583G, and F649S) required for the temperature-sensitive phenotype. Three of these changes (L21S, S226P, and F649S) were introduced individually and in various combinations into wild type SRB4, failing to elicit temperature sensitivity and confirming the requirement for additional amino acid changes (data not shown). The requirement for so many changes, distributed across the entire protein, suggests unfolding of the protein at restrictive temperatures, as opposed to an alternative such as temperature-dependent interaction with an accessory factor.

Consistent with instability of the mutant protein, isolation of Mediator from the srb4ts yeast strain was problematic. Several subunits, including Srb4, were lost during purification by the published procedure. We tried various approaches and arrived at a scheme involving affinity chromatography with an HA tag on the Med8 subunit. Maintenance of a low temperature and high protein concentration, and elution of the affinity column with both HA peptide and PreScission protease, proved crucial. Comparable yields of wild type and srb4ts Mediator, roughly 50% pure on the basis of silver stained SDS gels, were obtained (Fig. 6). Srb4 protein was present, although slightly substiochiometric, due to the instability noted above. Immunoblot analysis with antibodies against 18 Mediator subunits revealed all except Med6 in the srb4ts Mediator (Fig. 6). A band in the silver-stained gel at the position expected for Med15/Gal11 and Med5/Nut1 indicated the likely presence of these subunits as well.



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  		FIGURE 6.
Purified srb4ts Mediator. Eluates from HA affinity columns of wild type (wt) and srb4ts Mediators were analyzed by 10% SDS-PAGE, followed by silver staining (lanes 1 and 4) or by immunoblot analysis with antibodies against Mediator subunits (lanes 2 and 3).

 
Purified Mediator preparations were assayed for activity in transcription reconstituted with highly purified TBP, TFIIB, IIE, IIF, IIH, and pol II. Wild type and srb4ts Mediators stimulated basal transcription to about the same extent at 24 °C (Fig. 7, A and B). Stimulation in a fully purified system is alone indicative of a positive role of Mediator in basal transcription, apart from any anti-inhibitory effect. The activity of wild type Mediator was about 20% less at 30 °C, whereas that of srb4ts Mediator declined by about 70% at the elevated temperature (Fig. 7B). This demonstration of temperature-sensitive Mediator activity provides further evidence that the srb4ts phenotype is direct and not a secondary effect of diminished expression of another transcription factor.

Stimulation of basal transcription by srb4ts Mediator was less temperature-sensitive in the purified system than in crude extract (70% loss of activity in the purified system, compared with greater than 90% loss of activity in the extract). The mutant protein may be less stable in the extract, due to capture in the unfolded state by chaperonins or other interacting proteins. Or Mediator may perform additional roles in the crude extract, not required in the purified system, such as a role in chromatin remodeling (41).

The purified srb4ts Mediator supported activated transcription at 24 °C, although at about half the level of wild type Mediator (Fig. 7A). The slight defect may reflect the partial loss of subunits during purification. Adding back bacterially expressed Med6 failed to restore activity (data not shown), but other subunits may have been substoichiometric as well.

Finally, we examined the effect of the srb4ts mutation on the stimulation of TFIIH kinase activity. TFIIH is responsible for extensive phosphorylation of the CTD of pol II, required for the initiation of transcription (42). Diminished phosphorylation due to the srb4ts mutation could account for the mutant phenotype. The purified srb4ts Mediator, however, supported levels of CTD phosphorylation comparable with those of wild type Mediator at both 24 and 30 °C (Fig. 7C).



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  		FIGURE 7.
Purified srb4ts Mediator is temperature-sensitive for the stimulation of basal transcription but not for the stimulation of TFIIH kinase activity. A, transcription was performed with purified proteins at 24 °C as described under "Materials and Methods" with ~50, 100, or 200 ng of wild type or srb4ts Mediator as indicated, in the absence (lanes 1–3 and 7–9) or presence (lanes 4–6 and 10–12) of recombinant Gcn4 protein. Transcripts from templates containing Gcn4-binding sites (pGCN4) or Gal4-binding sites (pJJ470) and G-less cassettes (G–) are indicated on the right. 32P-Labeled {phi}X/Hinf III DNA molecular weight markers (M) are shown in the middle, with sizes on the left. B, transcription was performed as described for A at 24 or 30 °C as indicated. C, wild type or srb4ts Mediator (~50 ng in lanes 3, 5, 8, and 10 and 100 ng in lanes 4, 6, 8, and 11) was incubated with pol II (100 ng), TFIIH (15 ng), and {gamma}-32P-labeled ATP at 24 °C (lanes 1–6) or 30°C (lanes 7–11) as described under "Materials and Methods." In a control reaction, TFIIH was omitted (lane 1). CTD phosphorylation was revealed by SDS-PAGE and autoradiography. The band due to the Rpb1 subunit of pol II is indicated.

 
Mediator Does Not Reverse the Inhibition of Transcription In Vitro—Four subunits of the Not complex and two subunits of NC2 were identified in a screen for extragenic suppressors of the temperature-sensitive phenotype of the srb4ts mutation (32, 33). Both the Not complex and NC2 have been characterized as general negative regulators of transcription in vivo. We sought to observe opposing effects of these negative regulators and Mediator in vitro. We purified the nine-subunit Not complex from yeast by affinity chromatography with the use of a TAP tag. The subunits were identified by mass spectrometry and immunoblot analysis, and appeared approximately stoichiometric, with the exception of Caf130, which was present in a lesser amount (Fig. 8A). The purified complex failed to inhibit transcription either in crude extract or in the reconstituted system (Fig. 8, B and C); rather, it was slightly stimulatory, and its effect was roughly additive with that of Mediator, arguing against any interaction between the two complexes in vitro.

NC2 was expressed in bacteria and purified by affinity chromatography (Fig. 8D). The purified protein interfered with transcription in the reconstituted system, in keeping with results of others (32, 43), although a severalfold molar excess over the general transcription factors was required for greater than 50% inhibition (Fig. 8E). Addition of Mediator increased transcription about 3-fold at all concentrations of NC2; it did not restore transcription to the uninhibited level at low concentrations of NC2 (the curves in Fig. 8E do not converge at low NC2 concentration), as would have been expected if Mediator truly reversed the effect of NC2. Mediator evidently stimulated residual transcription, rather than opposing the effect of NC2.

Finally, we sought inhibitors in yeast cell extract whose effects could be reversed by Mediator. An srb4ts yeast extract was applied to Bio-Rex 70, and the resulting fractions were assayed for inhibition of basal transcription in srb4ts extract at 30 °C. The flow-through fraction was inhibitory, and inhibition was relieved by wild type Mediator (Fig. 9A), but not by srb4ts Mediator (data not shown). The Bio-Rex flow-through was further fractionated on DEAE-Sephacel and eluted at salt concentrations of 150, 300, and 600 mM. All fractions were inhibitory, and in the case of the 150 and 300 mM fractions, addition of Mediator was without effect. In the case of the 600 mM fraction (which does not contain NC2, Mot1, and Not complex as judged by immunoblotting), addition of Mediator elicited detectable transcription, but the extent of inhibition was the same at all Mediator concentrations (the curves in Fig. 9B do not converge at high Mediator concentration). So as with NC2, Mediator served to stimulate basal transcription rather than to reverse inhibition.



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  		FIGURE 8.
Purified Not complex does not inhibit transcription; inhibition by purified NC2 is not reversed by Mediator. A, eluate from calmodulin affinity column of Not complex was analyzed by 10% SDS-PAGE and stained with Coomassie Blue. Molecular weights are indicated on the left and subunits of Not complex on the right. B, transcription was performed as described forn Fig. 1 with wild type (wt)(lanes 1–3) or the srb4ts cell extract (lanes 4–6) in the presence or absence of purified Not complex (~50 or 100 ng). C, transcription was performed as in Fig. 7A in the presence or absence of 50 (+) or 100 (++) ng of purified Not complex. D, eluate from Hitrap Q column of recombiannt NC2 was analyzed by 10% SDS-PAGE and stained with Coomassie Blue. E, transcription was performed as described in the legend to Fig. 7A with amounts of recombinant NC2 indicated in the absence (lanes 1–7) or presence (lanes 7–12) of wild type Mediator. WCE, whole cell extract.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We (3) and others (14, 15) have previously demonstrated both the stimulation of basal transcription and support of activated transcription by Mediator in vitro. Genetic studies have shown the importance of Mediator for activated transcription in vivo. The physiological significance of the stimulation of basal transcription has remained an open question.



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  		FIGURE 9.
Inhibition of transcription by a partially purified yeast fraction is not reversed by Mediator. A, extract from srb4ts yeast (WCE(srb4ts)) was fractionated by chromatography on Bio-Rex 70 and on DEAE-Sephacel with elution by ammonium sulfate. Transcription was performed as in Fig. 7A with the addition of the Bio-Rex70 flow-through fraction (BR-FT) (0, 6.25, 12.5, 25, 50, and 25 µg, lanes 1–6), DEAE fractions (load (L), 10 µg, lanes 8 and 14; flow-through (FL), 10 µg, lanes 9 and 15; 150 mM ammonium sulfate eluate, 10 µg, lanes 10 and 16; 300 mM ammonium sulfate eluate, 10 µg, lanes 11 and 17; 600 mM ammonium sulfate eluate, 10 µg, lanes 12 and 18), and wild type (wt) Mediator (100 ng, lanes 6 and 13–18). B, transcription was performed as in Fig. 7A with the addition of DEAE-Sephacel 600 mM ammonium sulfate eluate, 5 µg) and wild type Mediator in the amounts indicated.

 
Our study of srb4ts Mediator addresses this question. We have found that both mutant cell extract and purified mutant Mediator are defective in basal transcription at an elevated temperature. The phenotype of the srb4ts mutant may therefore be ascribed to a defect in basal transcription. Mediator evidently performs a fundamental role in pol II transcription in vivo.

We note a discrepancy between the behavior of Mediator in vitro and in vivo. Whereas Mediator stimulates transcription in vitro, it is absolutely required in vivo (31). We suggest that the role of Mediator in transcription is the same in both cases, but the requirement for Mediator is relaxed in vitro, perhaps because of the high concentrations of transcription proteins used or because of the absence of histones or the like from the reconstituted reaction.

Three findings suggest that Mediator should be viewed as a general transcription factor. First, the action of Mediator is general. Virtually all pol II promoters are impaired in the srb4ts strain at a restrictive temperature. Second, the action of Mediator is positive. It stimulates transcription in a purified reconstituted system. It does not reverse inhibition by suppressors of the srb4ts mutation nor does there appear to be an as yet unidentified inhibitor of transcription whose effect is specifically opposed by Mediator. Third, the action of Mediator is required at the same time as that of the well known general transcription factors, immediately prior to the initiation of transcription (see also Ref. 44).

Several observations suggest that Mediator forms part of the preinitiation complex with general transcription factors, pol II, and promoter DNA. Mediator appears to interact with general transcription factors (44), remaining at the promoter along with TBP and TFIIB after initiation and the release of pol II, constituting a"re-initiation scaffold"for repeated rounds of transcription (45). Mediator also interacts directly with pol II, as mentioned above. Finally, electron microscopy and image processing have shown that Mediator binds along the backside of pol II, opposite the point of DNA entry, where TBP and TFIIB bind as well (46). This has led to the idea of a triple layer structure of the preinitiation complex, with Mediator on the outside, surrounding a general transcription factor-promoter DNA complex, which in turn envelops pol II (46).

Does Mediator stimulate transcription through its interactions with other components of the preinitiation complex, or is its effect primarily the reversal of inhibition by Not complex, NC2, or other factors, as suggested by genetic results (32, 33)? We have investigated this question by all means at our disposal, including the pursuit of as yet unidentified transcriptional inhibitors, and have found no evidence for the reversal of inhibition. We distinguish between two type of anti-inhibition; the stimulation of residual transcription, at whatever level, persists in the presence of inhibitor and true reversal of inhibition through specific antagonism of inhibitor action. Only in the case of true reversal of inhibition will Mediator be able to restore transcription to its original, uninhibited level, and this we have never observed.

The question thus remains of how Mediator stimulates transcription on its own, and to an even greater extent, in the presence of an activator protein. The simplest possibility is that Mediator stabilizes the preinitiation complex, speeding the assembly of the many components involved or enhancing the lifetime of the complex. Mediator might also influence the conformation of the complex, enhancing the rate of transcription initiation. There is currently little information to distinguish among such possibilities, but srb4ts Mediator and experiments of the sort reported here may help to do so. For example, the inactivation of Mediator by temperature jump as long as 10 min before the addition of NTPs did not interfere with transcription. This would be consistent with a role of Mediator in complex formation but not in subsequent initiation, or it could reflect a conformational effect of Mediator important for initiation that persists following the inactivation of Mediator. In another experiment reported here, an excess of all general transcription factors and pol II was added to srb4ts mutant cell extract. This failed to enhance transcription at the restrictive temperature, suggesting that complex formation was not limiting. Further work on these lines, clearly separating the events of complex formation and transcription initiation, will help unravel the Mediator mechanism.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM36659 (to R. D. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure. Back

1 To whom correspondence should be addressed. Tel.: 650-723-6988; Fax: 650-723-8464; E-mail: kornberg{at}stanford.edu.

2 The abbreviations used are: pol II, polymerase II; GST, glutathione S-transferase; HA, hemagglutinin; CTD, C-terminal domain. Back


   ACKNOWLEDGMENTS
 
We thank H. Komori for help making antibodies, P. Tempst and H E. Bromage for mass spectrometric analysis of purified Not complex, and G. Hartzog for technical advice. We thank B. Kemper for p{Delta}GCN4 DNA, G. Hartzog for 2xHA peptide and anti-HA antibodies, and D. Stillman, S. Bjorklund, C. Gustafsson, C. Denis, D. Auble, and M. Collart for antibodies.



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 DISCUSSION
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