The Soh1/MED31 Protein Is an Ancient Component of Schizosaccharomyces pombe and Saccharomyces cerevisiae Mediator

Abstract

We here demonstrated that the Soh1/MED31 protein is a stable component of Mediator complex isolated from Schizosaccharomyces pombe and Saccharomyces cerevisiae. Bioinformatic analysis traces the Soh1/MED31 family of Mediator subunits to the point of major eukaryotic divergence, before the appearance of the canonical heptapeptide repeat structure of the RNA polymerase II C-terminal domain. We here demonstrated that the Soh1/MED31 protein is a stable component of Mediator complex isolated from Schizosaccharomyces pombe and Saccharomyces cerevisiae. Bioinformatic analysis traces the Soh1/MED31 family of Mediator subunits to the point of major eukaryotic divergence, before the appearance of the canonical heptapeptide repeat structure of the RNA polymerase II C-terminal domain. The Mediator complex acts as a bridge, conveying regulatory information from enhancers and other control elements to the general transcription machinery. Mediator was originally identified in Saccharomyces cerevisiae and is required for the basal and regulated expression of nearly all RNA polymerase II (RNAP II) 1The abbreviations used are: RNAP II, RNA polymerase II; CTD, C-terminal domain; rpm, revolutions/min; TAP, tandem affinity purification; TEV, tobacco etch virus.-dependent genes (1Myers L.C. Kornberg R.D. Annu. Rev. Biochem. 2000; 69: 729-749Crossref PubMed Scopus (322) Google Scholar). Mediator-like complexes were later isolated from human cells (2Fondell J.D. Ge H. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8329-8333Crossref PubMed Scopus (468) Google Scholar, 3Sun X. Zhang Y. Cho H. Rickert P. Lees E. Lane W. Reinberg D. Mol. Cell. 1998; 2: 213-222Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 4Naar A.M. Beaurang P.A. Zhou S. Abraham S. Solomon W. Tjian R. Nature. 1999; 398: 828-832Crossref PubMed Scopus (376) Google Scholar, 5Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. Naar A.M. Erdjument-Bromage H. Tempst P. Freedman L.P. Nature. 1999; 398: 824-828Crossref PubMed Scopus (638) Google Scholar, 6Ryu S. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7137-7142Crossref PubMed Scopus (38) Google Scholar), mouse (7Jiang Y.W. Veschambre P. Erdjument-Bromage H. Tempst P. Conaway J.W. Conaway R.C. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8538-8543Crossref PubMed Scopus (275) Google Scholar), fission yeast (8Spahr H. Beve J. Larsson T. Bergstrom J. Karlsson K.A. Gustafsson C.M. J. Biol. Chem. 2000; 275: 1351-1356Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and fruit fly (9Park J.M. Gim B.S. Kim J.M. Yoon J.H. Kim H.S. Kang J.G. Kim Y.J. Mol. Cell. Biol. 2001; 21: 2312-2323Crossref PubMed Scopus (53) Google Scholar). Cross-species comparisons in several labs have led to the identification of metazoan counterparts for most yeast Mediator subunits (10Bourbon H.M. Aguilera A. Ansari A.Z. Asturias F.J. Berk A.J. Bjorklund S. Blackwell T.K. Borggrefe T. Carey M. Carlson M. Conaway J.W. Conaway R.C. Emmons S.W. Fondell J.D. Freedman L.P. Fukasawa T. Gustafsson C.M. Han M. He X. Herman P.K. Hinnebusch A.G. Holmberg S. Holstege F.C. Jaehning J.A. Kim Y.J. Kuras L. Leutz A. Lis J.T. Meisterernest M. Naar A.M. Nasmyth K. Parvin J.D. Ptashne M. Reinberg D. Ronne H. Sadowski I. Sakurai H. Sipiczki M. Sternberg P.W. Stillman D.J. Strich R. Struhl K. Svejstrup J.Q. Tuck S. Winston F. Roeder R.G. Kornberg R.D. Mol. Cell. 2004; 14: 553-557Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The Soh1/MED31 protein is a subunit of human (11Gu W. Malik S. Ito M. Yuan C.X. Fondell J.D. Zhang X. Martinez E. Qin J. Roeder R.G. Mol. Cell. 1999; 3: 97-108Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) and Drosophila Mediator (9Park J.M. Gim B.S. Kim J.M. Yoon J.H. Kim H.S. Kang J.G. Kim Y.J. Mol. Cell. Biol. 2001; 21: 2312-2323Crossref PubMed Scopus (53) Google Scholar). Although Soh1/MED31 orthologues are encoded by the S. cerevisiae and Schizosaccharomyces pombe genomes, these proteins have not been identified as Mediator components. The S. cerevisiae SOH1 gene was first identified as a suppressor of temperature-dependent growth of the hyper-recombination mutant hpr1Δ3 (12Fan H.Y. Klein H.L. Genetics. 1994; 137: 945-956Crossref PubMed Google Scholar). Later genetic studies linked SOH1 to components of the general transcription machinery (13Fan H.Y. Cheng K.K. Klein H.L. Genetics. 1996; 142: 749-759Crossref PubMed Google Scholar) and factors implicated in transcriptional elongation (14Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P. Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (527) Google Scholar, 15Malagon F. Tong A.H. Shafer B.K. Strathern J.N. Genetics. 2004; 166: 1215-1227Crossref PubMed Scopus (44) Google Scholar). A screen for septation mutants in S. pombe identified the sep10+ gene encoding a Soh1/MED31 orthologue (16Grallert A. Grallert B. Zilahi E. Szilagyi Z. Sipiczki M. Yeast. 1999; 15: 669-686Crossref PubMed Scopus (34) Google Scholar). The same screen also identified genes encoding S. pombe Mediator subunits Med8 and Pmc6/MED18. The null mutant of sep10+ causes sterility and hyphal growth in S. pombe (13Fan H.Y. Cheng K.K. Klein H.L. Genetics. 1996; 142: 749-759Crossref PubMed Google Scholar, 17Szilagyi Z. Grallert A. Nemeth N. Sipiczki M. Mol. Genet. Genomics. 2002; 268: 553-562Crossref PubMed Scopus (13) Google Scholar). We here report the identification of Soh1 and its orthologue Sep10 (here denoted spSoh1/MED31) as bona fide members of the S. cerevisiae and S. pombe Mediator complex. A thorough study of predicted Soh1/MED31 orthologues from a wide range of eukaryotic species revealed a highly conserved 69-amino acid motif. We used sequence analysis to trace the Soh1/MED31 family of Mediator subunits to the point of major eukaryotic divergence, before the appearance of the canonical heptapeptide repeat structure of the RNAP II C-terminal domain (CTD). Plasmid Construction—We generated the pFA6-2xFLAG-kanMX6 plasmid by introducing a double-stranded oligonucleotide linker (5′-CTC GAG ACT ATA AGG ATG ACG ATG ACA AGA TCG ACT ATA AGG ATG ACG ATG ACA AGT AGG GCC-3′; 5′-CGC GGG CCC TAC TTG TCA TCG TCA TCC TTA TAG TCG ATC TTG TCA TCG TCA TCC TTA TAG TCT CGA GAT-3′) between the AscI and PacI sites of pFA6a-GST-kanMX6, replacing the glutathione S-transferase (GST) tag with a tandem FLAG tag (18Longtine M.S. McKenzie A. II I Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4281) Google Scholar). We PCR-amplified the 3′-flanking region of SOH1 (forward primer, 5′-GCG CGC GAG CTC ACA CAC CTA ATT ATA ACT-3′; reverse, 5′-GCG CGC GAT ATC AGC TGC ATT GGT GAG GAT ATT-3′), and inserted the resulting DNA fragment between EcoRV and SacI of pFA6-2xFLAG-kanMX6, to generate pFA6-2xFLAG-dsSOH1. To construct pFA6-2xFLAG-SOH1wt, pFA6-2xFLAG-SOH1Δ1, pFA6-2xFLAG-SOH1Δ2, and pFA6-2xFLAG-SOH1Δ5, we PCR-amplified the coding region of SOH1 (or truncated versions thereof). We used primers 1 (forward, (5′-GCG CGC GGA TCC CCT CAA GCA CTA ATG GAA AC-3′) and 2 (reverse, (5′-GCG CGC CTC GAG AGT TGG CCC ATC TTT CTA C-3′) for SOH1wt; primers 3 (forward, (5′-CGC GCG GAT CCA GCT GAT GTC AAG CAC TAA TGG A-3′) and 4 (reverse, (5′-CGC GCG CTC GAG ACT GTT GAG TGA GTA AGT A-3′) for SOH1Δ1; and primers 3 (forward) and 6 (reverse, (5′-CGC GCG CTC GAG ACA TGA ACC CGT TCA ACA G-3′) for SOH1Δ2. The DNA fragments were inserted between the BamHI and XhoI sites of pFA6-2xFLAG-dsSOH1 to generate pFA6-2xFLAG-SOH1wt, pFA6-2xFLAG-SOH1Δ1, and pFA6-2xFLAG-SOH1Δ2. The pFA6-2xFLAG-SOH1Δ3, pFA6-2xFLAG-SOH1Δ4, and pFA6-2xFLAG-SOH1Δ5 constructs were made from two individual DNA fragments. We first PCR-amplified parts of the SOH1 coding region using primers 7 (forward, (5′-GCG CGG ATC CTA GAA TTC ATG TTG AAC GGG TTC ATG GAG-3′) and 2 (reverse) for SOH1Δ3; primers 8 (forward, (5′-GCG CGG ATC CTA GAA TTC ATG AGG TTC GAA GTG GAG CTC-3′) and 6 (reverse) for SOH1Δ4; and primers 8 (forward) and 2 (reverse) for SOH1Δ5. We next inserted the resulting DNA fragments between the BamHI and XhoI sites of pFA6-2xFLAG-dsSOH1. Finally, we PCR-amplified the 5′-flanking region of SOH1 using primers 9 (forward, (5′-GCG CGG ATC CAG CTG TCA GTA TGG CAT ACC-3′) and 10 (reverse, (5′-ATA TGA ATT CCT GGG TGG AGC TTG GGG-3′) and introduced the resulting DNA fragment between BamHI and EcoRI to generate the final pFA6-2xFLAG-SOH1Δ3, pFA6-2xFLAG-SOH1Δ4, and pFA6-2xFLAG-SOH1Δ5 constructs. Yeast Strains—Yeast strains used in this study are listed in Table I. Fission yeast Mediator was purified from a tandem affinity purification (TAP)-spMed7 strain (19Samuelsen C.O. Baraznenok V. Khorosjutina O. Spahr H. Kieselbach T. Holmberg S. Gustafsson C.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6422-6427Crossref PubMed Scopus (101) Google Scholar). Wild-type budding yeast Mediator was purified from a Med8-TAP strain kindly provided by Dr. Roger D. Kornberg (20Borggrefe T. Davis R. Bareket-Samish A. Kornberg R.D. J. Biol. Chem. 2001; 276: 47150-47153Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). For construction of S. cerevisiae strains expressing FLAG-tagged Soh1/MED31 variants, we digested the relevant pFA6-2xFLAG plasmids with PvuII and transformed Med8-TAP cells using the lithium acetate procedure (21Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2149) Google Scholar). Correct genomic integration was assayed by PCR using primers 9 (forward) and 11 (reverse, (5′-CCA GCG GTC GTA ATG-3′) and appeared as a 1.7-kb bandshift when compared with a nontransformed control.Table IStrains used in this studyStrainRelevant genotypeSourceS. pombeTP43h-, ade-M216, med7+::med7+-TAP-G418R19Samuelsen C.O. Baraznenok V. Khorosjutina O. Spahr H. Kieselbach T. Holmberg S. Gustafsson C.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6422-6427Crossref PubMed Scopus (101) Google ScholarS. cerevisiaeMed8-TAPMATa, pep4::HIS3/prb1::LEU2, prc1::HISG, can1, ade2, trp1, ura3, his3, leu2–3, 112, MED8::MED8-TAP-TRP20Borggrefe T. Davis R. Bareket-Samish A. Kornberg R.D. J. Biol. Chem. 2001; 276: 47150-47153Abstract Full Text Full Text PDF PubMed Scopus (56) Google ScholarCGC101MATa, pep4::HIS3/prb1::LEU2, prc1::HISG, can1, ade2, trp1, ura3, his3, leu2–3, 112, MED8::MED8-TAP-TRP, SOH1::SOH1-FLAG-G418RThis studyCGC103MATa, pep4::HIS3/prb1::LEU2, prc1::HISG, can1, ade2, trp1, ura3, his3, leu2–3, 112, MED8::MED8-TAP-TRP, SOH1::SOH1 1-FLAG-G418RThis studyCGC104MATa, pep4::HIS3/prb1::LEU2, prc1::HISG, can1, ade2, trp1, ura3, his3, leu2–3, 112, MED8::MED8-TAP-TRP, SOH1::SOH1 2-FLAG-G418RThis studyCGC105MATa, pep4::HIS3/prb1::LEU2, prc1::HISG, can1, ade2, trp1, ura3, his3, leu2–3, 112, MED8::MED8-TAP-TRP, SOH1::SOH1 3-FLAG-G418RThis studyCGC106MATa, pep4::HIS3/prb1::LEU2, prc1::HISG, can1, ade2, trp1, ura3, his3, leu2–3, 112, MED8::MED8-TAP-TRP, SOH1::SOH1 4-FLAG-G418RThis studyCGC107MATa, pep4::HIS3/prb1::LEU2, prc1::HISG, can1, ade2, trp1, ura3, his3, leu2–3, 112, MED8::MED8-TAP-TRP, SOH1::SOH1 5-FLAG-G418RThis study Open table in a new tab Protein Purification—S. cerevisiae cells were grown in YPD medium (10 g/liter yeast extract, 20 g/liter peptone, 20 g/liter glucose) to A600 3.5–4.0, and S. pombe cells were cultured to A600 2.0–2.5 in yeast extract supplement (YES) medium (5 g/liter yeast extract, 2 g/liter casamino acids, 20 g/liter glucose) supplemented with 0.2 g/liter adenine. For preparation of whole cell extract, we collected yeast cells from a 15-liter culture by centrifugation (JLA-10,500 rotor (Beckman Instruments), 9,500 rpm, 10 min, 4 °C), which were washed once with ice-cold water and suspended in 0.5 ml of 3× lysis buffer (200 mm KOH-Hepes, pH 7.8, 15 mm KCl, 1.5 mm MgCl2, 0.5 mm EDTA, 15% glycerol, 0.5 mm dithiothreitol, and protease inhibitors) per gram of cell pellet. For all purifications, we used a 100× stock of protease inhibitors containing 100 mm phenylmethylsulfonyl fluoride, 200 mm pepstatin, 60 mm leupeptin, and 200 mm benzamidine in 99.5% ethanol. Cells were lysed by bead beating (Bead-Beater, Stratech, London, UK) for 25 cycles, with each cycle consisting of 30 s of beating and 90 s of rest. We cleared the supernatant by centrifugation (JLA-10,500 rotor (Beckman Instruments) at 9,500 rpm, for 10 min, at 4 °C), added 1/9 vol of 2 m KCl, and stirred for 15 min. Polyethyleneimine was added to a final concentration of 0.02% followed by 15 min of stirring. Insoluble material was removed by ultracentrifugation (Ti45 rotor (Beckman Instruments), 42,000 rpm, 30 min, 4 °C). We purified Mediator according to the original TAP tag purification protocol (22Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2302) Google Scholar) with the following modifications. We added 900 μl of IgG-Sepharose (Amersham Biosciences) to 200 ml of extract and incubated for 2 h at 4 °C. The IgG-Sepharose was collected by centrifugation (JA-17 rotor (Beckman Instruments), 800 rpm, 1 min, 4 °C), loaded onto a Poly-Prep column (Bio-Rad), and washed with 100 ml of IgG wash buffer (20 mm HEPES-NaOH, pH 7.5, 150 mm NaCl, 0.1% IGEPAL CA-630). We eluted the Mediator in two steps: first, by incubation for 2 h at 16 °C with 200 units of tobacco etch virus (TEV) NIa protease (Invitrogen) in 2 ml of TEV buffer (10 mm HEPES-NaOH, pH 7.5, 150 mm NaCl, 0.5 mm EDTA, 0.1% IGEPAL CA-630, 1 mm dithiothreitol), which we designated IgG eluate 1, and second, by elution of the IgG-Sepharose by incubation with 8 m urea, 100 mm NaH2PO4, 10 mm Tris-Cl, pH 7.7, for 5 min at room temperature, which we called IgG eluate 2. For further purification of S. pombe Mediator, we loaded 500 μl of IgG eluate 1 onto a 1-ml heparin HiTrap column (Amersham Biosciences). The column was washed with 5 ml of buffer A-0.1 (20 mm HEPES-NaOH (pH 7.5), 10% glycerol, 0.5 mm EDTA, 1 mm dithiothreitol, protease inhibitors, and the molarity of sodium chloride indicated after the hyphen) and then developed with a linear gradient (12 ml) of A-0.1 to A-1.2. We purified S. cerevisiae Mediator further by loading 750 μl of IgG eluate 1 onto a 1-ml SP-Sepharose HiTrap column (Amersham Biosciences). The column was washed with 5 ml of buffer A-0.1 and developed with a linear gradient (12 ml) of A-0.1 to A-1.2. Immunoblotting and Production of Antibodies—Recombinant spSoh1/MED31 protein fused to GST was overproduced in Escherichia coli BL21 (DE3) pLysS cells (Stratagene) and subsequently purified using glutathione-Sepharose 4B (Amersham Biosciences) according to the supplier's recommendations. The purified GST-spSoh1/MED31 was used to immunize rabbits. The antisera used in this study were taken 10 days after the second booster injection (AgriSera, Vännäs, Sweden). We monitored Mediator with antibodies against Med7, Med8 (23Myers L.C. Gustafsson C.M. Bushnell D.A. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. Genes Dev. 1998; 12: 45-54Crossref PubMed Scopus (254) Google Scholar), spSrb4/MED17 (8Spahr H. Beve J. Larsson T. Bergstrom J. Karlsson K.A. Gustafsson C.M. J. Biol. Chem. 2000; 275: 1351-1356Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and Pmc3/MED27 (24Spahr H. Samuelsen C.O. Baraznenok V. Ernest I. Huylebroeck D. Remacle J.E. Samuelsson T. Kieselbach T. Holmberg S. Gustafsson C.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11985-11990Crossref PubMed Scopus (33) Google Scholar). Anti-Med1 antibodies were a gift from S. Bjorklund (Umeå University, Sweden), anti-Srb5/MED18 antibodies were a gift from R. A. Young for and antibodies were a gift from T. Fukasawa University, antibodies were from at the for for the Soh1/MED31 orthologues used are and The Soh1/MED31 sequence was kindly provided by the C. M. O. S. M. T. K. F. Y. Y. S. T. Y. T. A. M. H. K. H. F. S. S. S. T. Y. K. Y. Y. S. H. N. H. K. N. S. N. H. N. Y. T. Nature. 2004; PubMed Scopus Google Scholar). The Soh1/MED31 sequence protein was by the of and are provided for in this Protein were using J.D. F. F. PubMed Scopus Google Scholar) with were and with N. M. C. 1996; 12: Google Scholar). sequence were at the were at the G. J.M. 2004; 14: PubMed Scopus Google Scholar). Soh1/MED31 a of S. cerevisiae Soh1/MED31 was not identified as a Mediator subunit in of the S. cerevisiae complex. We have the Soh1/MED31 the used in analysis have to proteins D.J. 1999; PubMed Scopus Google Scholar). We to the of Soh1/MED31 in the S. cerevisiae We used a yeast strain with a TAP tag fused with the Med8 Mediator subunit and introduced a tandem FLAG tag at the of the Soh1/MED31 protein We purified Mediator IgG-Sepharose and of with Mediator subunits We purified Mediator further SP-Sepharose and of with Mediator subunit We that Soh1/MED31 is a subunit of the S. cerevisiae Mediator complex. spSoh1/MED31 a of the S. pombe the spSoh1/MED31 protein is a subunit of S. pombe Mediator, we a against protein in S. pombe Mediator with a C-terminal TAP tag with the gene was purified IgG-Sepharose and We used analysis and of spSoh1/MED31 with Mediator subunits Pmc3/MED27 and spSrb4/MED17 We purified S. pombe Mediator a and used to the which and We at to the predicted of for acid of the spSoh1/MED31 protein not We that spSoh1/MED31 is a subunit of the S. pombe The SOH1 of Mediator complexes purified to are from and which are the eukaryotic J.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We to analysis of the Soh1/MED31 family by using from a wide of with of sequence Z. Grallert A. Nemeth N. Sipiczki M. Mol. Genet. Genomics. 2002; 268: 553-562Crossref PubMed Scopus (13) Google Scholar). against and sequence tag we highly Soh1/MED31 orthologues in other of the eukaryotic and several We for further analysis using of and these using the sequence J.D. F. F. PubMed Scopus Google Scholar). that were were from the analysis to We that all sequence a acid which a of conserved and is in and using the Soh1/MED31 not between eukaryotic The two mutant for in S. cerevisiae and in S. are by the Soh1 as indicated in 3 (12Fan H.Y. Klein H.L. Genetics. 1994; 137: 945-956Crossref PubMed Google Scholar). The Soh1 for with S. cerevisiae the of the Soh1 for Mediator we generated a of S. cerevisiae in which the SOH1 gene was by mutant versions fused with a FLAG tag encoding the DNA fragment Mediator was purified from the and Soh1/MED31 was with against the FLAG tag We not Soh1/MED31 by in protein transcription PCR indicated of expression for the mutant constructs not Soh1/MED31 was from Mediator when we the Soh1 a of the mutant (12Fan H.Y. Klein H.L. Genetics. 1994; 137: 945-956Crossref PubMed Google Scholar), at in the of the Soh1 to with a of the in which the region the Soh1 been with Mediator, and we also Mediator of a C-terminal mutant the Soh1 is required for stable with Mediator, is not we the Soh1 in we with the Mediator complex. We here report that Soh1/MED31 is a stable component of Mediator isolated from S. pombe and S. cerevisiae. studies in yeast and the of Soh1/MED31 in metazoan Mediator that the of Soh1/MED31 with S. pombe and S. cerevisiae Mediator is relevant and not of We that to Soh1/MED31 in of yeast Mediator complexes (8Spahr H. Beve J. Larsson T. Bergstrom J. Karlsson K.A. Gustafsson C.M. J. Biol. Chem. 2000; 275: 1351-1356Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, L.C. Gustafsson C.M. Bushnell D.A. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. Genes Dev. 1998; 12: 45-54Crossref PubMed Scopus (254) Google Scholar, Y.J. Bjorklund S. Y. Kornberg R.D. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, C.M. L.C. Beve J. Spahr H. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) was of the of in to proteins D.J. 1999; PubMed Scopus Google Scholar). this we also Mediator subunit and demonstrated that Soh1/MED31 is conserved in The of Soh1/MED31 the of the eukaryotic transcription machinery. The of RNAP II a in the of of the and of have led to the that canonical are conserved in a of eukaryotic and which complex of J.W. B.D. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). to this the control RNAP II transcription by was in the of of gene expression that are a of complex eukaryotic J.W. B.D. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). The is also for Mediator and a of Mediator is its to (23Myers L.C. Gustafsson C.M. Bushnell D.A. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. Genes Dev. 1998; 12: 45-54Crossref PubMed Scopus (254) Google Scholar, Y.J. Bjorklund S. Y. Kornberg R.D. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). The Mediator complex with (23Myers L.C. Gustafsson C.M. Bushnell D.A. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. Genes Dev. 1998; 12: 45-54Crossref PubMed Scopus (254) Google Scholar, Y.J. Bjorklund S. Y. Kornberg R.D. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar), and the genes encoding for Mediator subunits were first identified in a screen for which the of a mutant Young R.A. Genetics. PubMed Google Scholar). We were to conserved Soh1/MED31 orthologues in G. and P. which the canonical repeat structure J.W. B.D. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). The that Mediator-like in from and to the RNAP II in that before the of the canonical heptapeptide Finally, analysis with Soh1/MED31 we not sequence of Soh1/MED31 to RNA as (12Fan H.Y. Klein H.L. Genetics. 1994; 137: 945-956Crossref PubMed Google Scholar). The original of the of the for that was the The of the of the is the of the which grown the first in at the that is a subunit of S. cerevisiae Mediator N. H. F. and M. We are for from the C. and the of We and for