Identification of five new mediator subunits

A highly purified mediator fraction was resolved by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF)-membrane, and stained with Ponceau S. Bands denoted Med2, Med3, Med4, Med5, Med7, and Med8 (see Fig. ​Fig.2)2) were excised and digested with trypsin, and the resulting peptides were fractionated by reverse-phase high-pressure liquid chromatography (HPLC) and sequenced. Perfect matches were found to sequences in the Saccharomyces cerevisiae genome database (Table ​(Table1),1), and the masses of peptides determined by mass spectrometry were in good agreement with the theoretical masses of the expected tryptic peptides for the individual gene products.

Table 1

Mediator subunits

Gene	Deletion phenotype	Human homolog	Protein mass (kD)	Apparent mass (kD)a	pI	Referenceb (yeast ORF)
GAL11	conditional		120	130	10.0	(1)
RGR1	inviable	+	123	115	9.0	(1)
SIN4	conditional		111	100	8.8	(1)
SRB4	inviable		78	98	5.1	(2)
MED1	conditional		64	70	4.7	(3)
MED2	conditional		48	62	4.4	this study (YDL005c)
PGD1c	conditional		47	48	9.4	this study
MED4	inviable		32	38d	4.5	this study (YOR174w)
MED6	inviable	+	32	36d	5.2	(4)
SRB5	conditional		34	35	4.7	(2)
MED7	inviable	+	32	31	6.7	this study (YOL135c)
MED8	inviable		25	30	5.3	this study (YBR193c)
ROX3	inviable		25	30	6.6	(5)
SRB2	conditional		23	27	5.2	(2)
SRB7	inviable	+	16	19	4.8	(6)
SRB6	inviable		14	15	4.6	(2)

Open in a separate window

Boldface type indicates the five new mediator subunits reported in this paper. 

^aAs judged by SDS-PAGE analysis with respect to molecular weight standards. 

^b(1) Li et al. (1995); (2) Thompson et al. (1993); (3) Bjorklund et al. (unpubl.); (4) Lee et al. (1997); (5) Gustafsson et al. (1997); (6) Hengartner et al. (1995). 

^cThis gene is also known as HRS1. 

^dMed4 and Med6 are often observed as doublets. 

Med4 and Med5 yielded peptide sequences from the same uncharacterized open reading frame, and thus derived from a single protein migrating as a doublet, hereafter referred to as Med4. The final member of the Gal11 module, the p50/Med3 protein, proved to be the product of the previously identified PGD1 (HRS1) gene (Bröhl et al. 1994). Med7 derived from an uncharacterized open reading frame. The Med8 band gave rise to peptide sequences from two open reading frames, one corresponding to the previously identified holoenzyme component Rox3 (Gustafsson et al. 1997), and a second, equally abundant protein, from an open reading frame partially characterized in the yeast chromosome II sequencing effort (Démolis et al. 1993), for which we retain the designation Med8.

Although none of the MED sequences show enzymatic motifs or other strong homologies clearly related to mediator function in transcription, there are some features of note in the newly defined sequences. The existence of a putative leucine zipper motif in MED8 at amino acids 69–90 has been recognized previously (Démolis et al. 1993). MED2 contains an asparagine-rich region at the carboxy terminus of >100 amino acids. A polyglutamine stretch, common to many transcription proteins, including Gal11, has been noted in Pgd1(Hrs1) (Santos-Rosa et al. 1996). Along with other members of the Gal11 module, Pgd1(Hrs1) is basic, whereas Med7 and Rox3 are neutral, and the rest of the mediator subunits are acidic (Table ​(Table11).

Med2, Med4, Med7, and Med8 were expressed in recombinant form, and polyclonal antibodies were obtained. The association of these proteins with RNA polymerase II holoenzyme was shown by copurification and coimmunoprecipitation. RNA polymerase II holoenzyme was purified from strain BJ926 by chromatography on Bio-Rex 70, DEAE–Sephacel, hydroxyapatite, Mono Q, and Bio-Sil SEC 400. Immunoblot analysis showed coelution from hydroxyapatite and Bio-Sil SEC 400 of Med2, Pgd1(Hrs1), Med4, Med7, Med8, the polymerase subunits Rpb1 and Rpb3, and Srb4 (Fig. ​(Fig.4).4).

Open in a separate window

Open in a separate window

Figure 4

Med proteins comigrate during hydroxyapatite chromatography and gel filtration. (A) The peak of RNA polymerase II holopolymerase from DEAE-Sephacel was applied to a 20 ml hydroxyapatite column and eluted with a 200 ml gradient of 0.01–0.2 mm potassium phosphate. Fractions were analyzed by SDS-PAGE in a 10% gel and immunoblotted with antibodies directed against the proteins indicated. (B) The mediator peak from Mono Q was subjected to gel filtration through Bio-Sil SEC 400. Fractions were analyzed by SDS-PAGE in a 10% gel and immunoblotted with antibodies directed against the proteins indicated.

Immunoprecipitation was performed with anti-Med2 antibodies coupled to protein A–Sepharose. Holopolymerase was incubated with the antibody–Sepharose, followed by washing under stringent conditions. Immunoblot analyses revealed that Med2, Pgd1(Hrs1), Med4, Med7, Med8, Rpb3, and Srb4 were almost entirely bound by the antibody–Sepharose (Fig. ​(Fig.5A).5A). Immunoprecipitation with anti-Med4 antibodies gave the same result (Fig. ​(Fig.5B).5B). We conclude that Med2, Pgd1(Hrs1), Med4, Med7, and Med8 in the holopolymerase preparation were entirely associated with the holoenzyme.

Open in a separate window

Figure 5

Co-immunoprecipitation of Med proteins with RNA polymerase II holoenzyme. (A) Immunoprecipitation by anti-Med2 antibody coupled to protein A–Sepharose. An RNA polymerase II holopolymerase fraction from Mono Q (Load, 5 μg) was incubated with anti-Med2 antibody beads. The supernatant (Sup) was removed, the beads were washed, and immunoprecipitated protein was eluted (Pel). Equal amounts of the load, supernatant, and eluted proteins were analyzed by immunoblotting with antibodies against the proteins indicated. (B) Immunoprecipitation with anti-Med4 antibody coupled to protein A–Sepharose, as in A.

MED gene requirement for cell viability

Yeast strains were constructed in which each of the previously uncharacterized open reading frames encoding Med proteins was deleted. Following sporulation and tetrad dissection, viability segregated 2:2 for the med4, med7, and med8 deletions. Tetrad dissection of the med2 deletion strain yielded four viable spores, but the two spores containing the disrupted gene were significantly smaller and unable to grow on galactose as the sole carbon source (data not shown).

Mediator binds directly to a CTD peptide

A GST fusion protein that included all 26 repeats of the yeast CTD and 40 amino acids of linker sequence was loaded on glutathione–Sepharose. Pure mediator was completely bound by the resulting GST–CTD Sepharose, whereas none was bound by a GST–Sepharose control (Fig. ​(Fig.6).6). What little mediator was not bound by the GST–CTD Sepharose could most likely be attributed to a small amount of GST–CTD that bled off the column. Approximately half of the bound mediator was eluted with glutathione, which also displaced a majority of the GST–CTD. The remaining mediator and GST–CTD were eluted under denaturing conditions. There was no mediator in eluates from the GST–Sepharose control.

Open in a separate window

Figure 6

Mediator binds directly to the CTD. Free mediator was applied to GST–CTD and GST (control) resins and eluted with glutathione (Elution 1) and SDS (Elution 2). Proteins were analyzed by immunoblotting with antibodies directed against Med4, Med8, and the CTD (to reveal the presence of GST–CTD).

CTD required for mediator function in vitro

RNA polymerase II lacking a CTD (Pol II ΔCTD) was prepared by proteolysis as described (Li and Kornberg 1994). Pol II ΔCTD was indistinguishable from wild-type enzyme in a basal transcription reaction (Fig. ​(Fig.7).7). Mediator, however, failed to stimulate basal transcription by Pol II ΔCTD, nor did it elicit a response of Pol II ΔCTD to Gal4–VP16 or GCN4, in marked contrast to its effect on the wild-type polymerase (Fig. ​(Fig.3A;3A; Kim et al. 1994).

Open in a separate window

Figure 7

CTD requirement for mediator function in vitro. Transcription was performed as described with wild type polymerase (1 μg) or Pol II ΔCTD (1 μg) in the presence of mediator (500 ng), Gal4–VP16 (10 ng), and GCN4 (10 ng) as indicated.

Protein purification

Fractionation of whole cell extract by chromatography on Bio-Rex 70 (Bio-Rad), DEAE–Sephacel (Pharmacia), Bio-Gel HTP hydroxyapatite (Bio-Rad), and Mono Q was as described (Li et al. 1996) with the following modifications. Active dry yeast (2.6 kg) collected before drying (Fleischmann’s, Oakland, CA) was suspended in lysis buffer and the cells were disrupted as described (Kim et al. 1994). After fractionation on hydroxyapatite, the mediator/holopolymerase peak, detected by immunoblotting, was loaded on a Mono Q 10/10 column (Pharmacia). After washing with 40 ml of buffer Q-0.15 and 40 ml of Q-0.5, the column was developed with a linear gradient (112 ml) of buffer Q-0.5 to Q-1.2. Immunoblotting revealed a peak of free mediator eluting around Q-0.6 and a peak of holopolymerase eluting around Q-0.8. The peak of free mediator was pooled and dialyzed against H-0.1 [containing 20 mm potassium phosphate (pH 7.6), 10% glycerol, 0.2 mm EDTA, 1 mm DTT, protease inhibitors, and the molarity of potassium acetate indicated after the hyphen] for 4 hr. The dialysate was diluted with H-0 to the conductivity of H-0.1 and applied to a TSK–Heparin–5-PW HPLC column (3.3 ml, 75×7.5 mm, Supelco) equilibrated in H-0.1. After washing with two column volumes of H-0.1 and 4 column volumes of H-0.25, the column was developed with a linear gradient (33 ml) of H-0.25—H-0.75 at a flow rate of 0.5 ml/min, and fractions of 0.75 ml were collected. The peak of mediator was at ~H-0.4.

Peak mediator fractions were dialyzed against buffer A-0 [containing 20 mm HEPES-KOH (pH 7.6), 10% glycerol, 1 mm EDTA, 1 mm DTT, protease inhibitors, and the molarity of potassium acetate indicated after the hyphen], adjusted to a conductivity of A-0.1, and applied to a Mono S 5/5 FPLC column (Pharmacia) equilibrated in A-0.1. After washing with five column volumes of A-0.1 and 5 column volumes of A-0.2, the column was developed with a linear gradient (12 ml) of A-0.2–A-1.0 at a flow rate of 0.5 ml/min, and fractions of 0.35 ml were collected. The peak of mediator was at ~A-0.5. To test the homogeneity of the multiprotein complex, a portion (0.25 ml) of the Mono S peak mediator fraction was applied to a Bio-Sil SEC 400 HPLC column (Bio-Rad) equilibrated in A-0.5.

RNA polymerase II holoenzyme was purified from 650 grams of S. cerevisiae strain BJ926 as described in Li et al. (1996).

Protein sequencing

Peptides were generated from Med2, Med3, Med4, Med7, and Med8 proteins bound to PVDF by tryptic digestion in situ (Erdjument-Bromage et al. 1994; Lui et al. 1996) and were fractionated by reversed-phase HPLC (Elicone et al. 1994) with the use of a 1-mm Reliasil C₁₈ column. Selected peak fractions were analyzed by a combination of automated Edman chemical degradation (Tempst et al. 1994) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. Peptide sequences were compared with entries in the Saccharomyces Genome Database (SGD, Stanford Genomic Resources, Stanford University) with the use of the National Center for Biotechnology Information (NCBI) BLAST program. Experimental masses of these and additional peptides were compared with the theoretical average isotopic masses of fragments expected to result from tryptic digestion of the identified proteins (with the use of PeptideSearch software; Dr. Matthias Mann, European Molecular Biology Laboratory, Heidelberg, Germany).

Antibodies and immunoblot analyses

Recombinant Med2, Med4, Med7, and Med8 proteins fused to glutathione S-transferase (Table ​(Table3)3) were overproduced in Escherichia coli BL21(DE3) pLysS cells and purified from inclusion bodies as described (Cairns et al. 1994). For each of the GST–Med fusions, ~5 mg of protein was separated by SDS-PAGE and used to immunize rabbits. The antisera used in this study were taken 10 days after the second booster injection (BabCO, Berkeley, CA). Anti-HRS1 antibodies (Santos-Rosa et al. 1996) were a gift from A. Aguilera (Universidad de Sevilla, Spain). Anti-RPB3 antibodies were a gift from R. Burgess and N. Thompson (McArdle Laboratory for Cancer Research, Madison, WI).

Immunoprecipitation

Anti-Med2 and anti-Med4 antibodies were coupled to protein A-Sepharose beads and were used to immunoprecipitate a purified holopolymerase as described (Li et al. 1995), with the following modifications. Prior to incubation with the holopolymerase fraction, 50 μl of the anti-Med6 beads were washed with 100 μl of 50 mm glycine (pH 2.3), 150 mm NaCl, followed by several washes with 100 μl of IP-200 wash buffer (20 mm HEPES at pH 7.6, 10% glycerol, 12.5 mm MgCl₂, 0.1 mm EDTA, 0.2% NP-40, 0.1 mm DTT, 200 mm potassium acetate). A Mono Q fraction of holopolymerase (10 μl) was diluted with 40 μl of buffer Q-0.5 (no dithiothreitol or protease inhibitors), centrifuged for 5 min at 13,000 rpm, and incubated with the beads for 4 hr at 4°C. The beads were then washed three times with 200 μl of buffer IP-200 and eluted twice with 17.5 μl of 5 m urea (10 min at room temperature). The eluates combined with 20 μl of 2× SDS gel-loading buffer were loaded on gels for immunoblotting.

Transcription and CTD phosphorylation assays

Transcription and CTD phosphorylation assays were performed as described (Kim et al. 1994), with the following modifications. Pure TFIIF (Henry et al. 1992) was added to all transcription reactions, and TFIIH was purified as described by Svejstrup et al. (1994). All transcription reactions were performed at a final potassium acetate concentration of 180 mm.

Mediator–CTD interaction

To construct a GST–CTD expression construct the carboxy-terminal domain of Rpb1 was amplified by PCR, by use of the primers 5′-GCGCGCGGATCCCCAATGACGCTATGGCTGGAGG-3′ and 5′-CCGGCCGAATTCTTATCATCTGGAATTTTCATTTTCAT-3′. The PCR product was then cleaved with EcoRI and BamHI, and inserted into pGEX-3X (Pharmacia). Pure GST–CTD and GST (30 μg) were bound to 25 μl of Glutathione Sepharose 4B (Pharmacia) following the manufacturer’s instructions. The GST–CTD and GST resins were incubated with 1 μg of pure mediator diluted in 100 μl of buffer T-0.3 [buffer A containing 20 mm Tris-Acetate (pH 7.8), 10% glycerol, 0.2 mm EDTA, 0.01 % NP-40, 1 mm DTT, and the molarity of potassium acetate indicated after the hyphen] for 2 hr at 4°C with gentle agitation, followed by three washes with buffer T-0.2 plus 0.1% NP-40. The resins were eluted first with 10 mm reduced glutathione, 50 mm Tris-acetate (pH 7.8) and then by boiling in SDS-PAGE buffer. The load, supernatant, washes, and elutes were analyzed by SDS-PAGE and immunoblotting.

We thank G. Jensen for purified transcription proteins and B. Cairns for much help in the analysis of the Δmed2 phenotype. L.C.M. was a recipient of a Cancer Research Fund of the Damon Runyon–Walter Winchell Foundation Fellowship, DRG-1361. C.M.G. was a recipient of a Swedish Cancer Society postdoctoral fellowship. This research was supported by National Institutes of Health grant GM36659 to R.D.K., by National Science Foundation grant DBI-940123 to P.T., and by National Cancer Institutes core grant 5 P30 CA08748 to the Sloan-Kettering Sequencing Laboratory.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.