Med19(Rox3) Regulates Intermodule Interactions in the Saccharomyces cerevisiae Mediator Complex
2006; Elsevier BV; Volume: 282; Issue: 8 Linguagem: Inglês
10.1074/jbc.m609484200
ISSN1083-351X
AutoresShamara Baidoobonso, Benjamin W. Guidi, Lawrence C. Myers,
Tópico(s)RNA Research and Splicing
ResumoThe Saccharomyces cerevisiae Mediator is a 25-subunit complex that facilitates both transcriptional activation and repression. Structural and functional studies have divided Mediator subunits into four distinct modules. The Head, Middle, and Tail modules form the core functional Mediator complex, whereas a fourth, the Cyc-C module, is variably associated with the core. By purifying Mediator from a strain lacking the Med19(Rox3) subunit, we have found that a complex missing only the Med19(Rox3) subunit can be isolated under mild conditions. Additionally, we have established that the entire Middle module is released when the Δmed19(rox3) Mediator is purified under more stringent conditions. In contrast to most models of the modular structure of Mediator, we show that release of the Middle module in the Δmed19(rox3) Mediator leaves a stable complex made up solely of Head and Tail subunits. Both the intact and Head-Tail Δmed19(rox3) Mediator complexes have defects in enhanced basal transcription, enhanced TFIIH phosphorylation of the CTD, as well as binding of RNA Pol II and the CTD. The largely intact Δmed19(rox3) complex facilitates activated transcription at levels similar to the wild type Mediator. In the absence of the Middle module, however, the Δmed19(rox3) Mediator is unable to facilitate activated transcription. Although the Middle module is unnecessary for holding the Head and Tail modules together, it is required for the complex to function as a conduit between activators and the core transcription machinery. The Saccharomyces cerevisiae Mediator is a 25-subunit complex that facilitates both transcriptional activation and repression. Structural and functional studies have divided Mediator subunits into four distinct modules. The Head, Middle, and Tail modules form the core functional Mediator complex, whereas a fourth, the Cyc-C module, is variably associated with the core. By purifying Mediator from a strain lacking the Med19(Rox3) subunit, we have found that a complex missing only the Med19(Rox3) subunit can be isolated under mild conditions. Additionally, we have established that the entire Middle module is released when the Δmed19(rox3) Mediator is purified under more stringent conditions. In contrast to most models of the modular structure of Mediator, we show that release of the Middle module in the Δmed19(rox3) Mediator leaves a stable complex made up solely of Head and Tail subunits. Both the intact and Head-Tail Δmed19(rox3) Mediator complexes have defects in enhanced basal transcription, enhanced TFIIH phosphorylation of the CTD, as well as binding of RNA Pol II and the CTD. The largely intact Δmed19(rox3) complex facilitates activated transcription at levels similar to the wild type Mediator. In the absence of the Middle module, however, the Δmed19(rox3) Mediator is unable to facilitate activated transcription. Although the Middle module is unnecessary for holding the Head and Tail modules together, it is required for the complex to function as a conduit between activators and the core transcription machinery. The Mediator complex is a conserved interface between gene-specific regulatory proteins and the general transcription apparatus of eukaryotes at transcription initiation (1Kornberg R.D. Trends Biochem. Sci. 2005; 30: 235-239Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Saccharomyces cerevisiae Mediator is comprised of 25 subunits (2Bjorklund S. Gustafsson C.M. Trends Biochem. Sci. 2005; 30: 240-244Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Structural (3Chadick J.Z. Asturias F.J. Trends Biochem. Sci. 2005; 30: 264-271Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), biochemical (2Bjorklund S. Gustafsson C.M. Trends Biochem. Sci. 2005; 30: 240-244Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), genetic (4Myers L.C. Kornberg R.D. Annu. Rev. Biochem. 2000; 69: 729-749Crossref PubMed Scopus (321) Google Scholar), and genomic (5van de Peppel J. Kettelarij N. van Bakel H. Kockelkorn T.T. van Leenen D. Holstege F.C. Mol. Cell. 2005; 19: 511-522Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) studies have enabled the provisional assignment of yeast Mediator subunits into four distinct modules. Each of these modules is thought to have a unique role in the structure and function of the entire complex. The core functional Mediator complex was initially defined as the minimal intact complex, purified from a wild type strain, capable of facilitating activated transcription in a system reconstituted from highly purified general transcription factors (6Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (886) Google Scholar, 7Myers 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 (252) Google Scholar). Single-particle electron microscopic analysis of core Mediator identified three areas of density that were referred to as the Tail, Middle, and Head modules of the complex (3Chadick J.Z. Asturias F.J. Trends Biochem. Sci. 2005; 30: 264-271Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). A fourth module, called the Cyc-C module, is found to be variably associated with the core Mediator modules and may regulate its function (8Samuelsen 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 (98) Google Scholar). The modular structure of the yeast complex appears to be conserved in Mediator complexes purified from metazoan cells (9Malik S. Roeder R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 10Conaway R.C. Sato S. Tomomori-Sato C. Yao T. Conaway J.W. Trends Biochem. Sci. 2005; 30: 250-255Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Biochemical and genetic studies have helped assemble a reasonable model for the composition of, and the interactions within, the structural modules of Mediator (summarized in Refs. 3Chadick J.Z. Asturias F.J. Trends Biochem. Sci. 2005; 30: 264-271Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar and 11Guglielmi B. van Berkum N.L. Klapholz B. Bijma T. Boube M. Boschiero C. Bourbon H.M. Holstege F.C. Werner M. Nucleic Acids Res. 2004; 32: 5379-5391Crossref PubMed Scopus (174) Google Scholar; see Fig. 7A). Little, however, is known about how these modules are held together, what the functional implications of these intermodular interactions are, and how these interactions are regulated. Genetic, genomic, and functional assays in vitro have provided support for the model of the modular structure of Mediator but also raised further questions on how the modules function together. Mediator, purified from wild type cells, has three activities in vitro: the ability to facilitate activated transcription, the ability to enhance basal transcription, and the ability to enhance phosphorylation of the C-terminal domain (CTD) 2The abbreviations used are: CTD, C-terminal domain; Pol, polymerase; GST, glutathione S-transferase. of RNA Pol II by TFIIH (7Myers 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 (252) Google Scholar). Mutations in the Tail module subunits of Mediator generally lead to defects in activated transcription but have little effect on enhancement of basal transcription or CTD phosphorylation (12Myers L.C. Gustafsson C.M. Hayashibara K.C. Brown P.O. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 67-72Crossref PubMed Scopus (153) Google Scholar, 13Lee Y.C. Park J.M. Min S. Han S.J. Kim Y.J. Mol. Cell. Biol. 1999; 19: 2967-2976Crossref PubMed Scopus (133) Google Scholar). Mutations in the Head module proteins have broad effects on all three functions in vitro (13Lee Y.C. Park J.M. Min S. Han S.J. Kim Y.J. Mol. Cell. Biol. 1999; 19: 2967-2976Crossref PubMed Scopus (133) Google Scholar, 14Lee Y.C. Kim Y.J. Mol. Cell Biol. 1998; 18: 5364-5370Crossref PubMed Scopus (70) Google Scholar). Middle module mutant Mediators, Δmed9 and med10ts, appear to have defects in basal and activated transcription (15Han S.J. Lee Y.C. Gim B.S. Ryu G.H. Park S.J. Lane W.S. Kim Y.J. Mol. Cell Biol. 1999; 19: 979-988Crossref PubMed Scopus (74) Google Scholar), although the structural composition of these complexes has not been evaluated. A recent genomic study and a wealth of data from elegant yeast genetic screens have outlined key distinctions between the subunits in the Middle module and those in the Tail and the Head modules of Mediator. The Tail module subunits are largely encoded by nonessential genes, whereas the Middle and Head domains are encoded by roughly equal numbers of essential and nonessential genes. Genetic studies of various regulatory systems in yeast have revealed that subunits in the Tail and Head module have a positive influence on gene expression (4Myers L.C. Kornberg R.D. Annu. Rev. Biochem. 2000; 69: 729-749Crossref PubMed Scopus (321) Google Scholar). In contrast, a number of genetic screens have shown that mutations in Middle, Cyc-C, and some Tail module subunits lead to derepression of heat shock factor basal transcription (16Singh H. Erkine A.M. Kremer S.B. Duttweiler H.M. Davis D.A. Iqbal J. Gross R.R. Gross D.S. Genetics. 2006; 172: 2169-2184Crossref PubMed Scopus (39) Google Scholar), maltose-induced genes (17Wang X. Michels C.A. Genetics. 2004; 168: 747-757Crossref PubMed Scopus (19) Google Scholar), glucose-repressed genes (18Song W. Treich I. Qian N. Kuchin S. Carlson M. Mol. Cell Biol. 1996; 16: 115-120Crossref PubMed Scopus (110) Google Scholar), and the HO gene (19Tabtiang R.K. Herskowitz I. Mol. Cell Biol. 1998; 18: 4707-4718Crossref PubMed Scopus (45) Google Scholar, 20Jiang Y.W. Dohrmann P.R. Stillman D.J. Genetics. 1995; 140: 47-54Crossref PubMed Google Scholar). Systematic gene expression microarray and clustering analyses of Mediator mutant strains found a correlation pattern among subunits of the complex (5van de Peppel J. Kettelarij N. van Bakel H. Kockelkorn T.T. van Leenen D. Holstege F.C. Mol. Cell. 2005; 19: 511-522Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). A strong positive correlation was observed between members of Head and Tail modules, whereas a negative correlation was observed between members of these modules and the subunits of the Middle module. Given the separation of the Head and Tail modules by the Middle module in the structural models, it is an open question how the Head and Tail could function in concert with each other to exert a positive effect on gene expression on select sets of genes. Moreover, it is unclear how Middle module defects could lead to up-regulation of transcription, whereas these subunits are seemingly required to link two positive acting modules of the complex. A Mediator subunit that plays a key role in both activation and repression is Med19(Rox3). Med19(Rox3) was originally identified in a search for mutants increasing aerobic expression of the CYC7 gene (21Rosenblum-Vos L.S. Rhodes L. Evangelista Jr., C.C. Boayke K.A. Zitomer R.S. Mol. Cell Biol. 1991; 11: 5639-5647Crossref PubMed Scopus (67) Google Scholar). Analysis of purified Mediator by mass spectrometry conclusively demonstrated that Med19(Rox3) was a subunit of Mediator (22Gustafsson C.M. Myers L.C. Li Y. Redd M.J. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. J. Biol. Chem. 1997; 272: 48-50Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Med19(Rox3) was initially found to be encoded by an essential gene product; however, a strain made as part of the genome wide deletion library lacks MED19(ROX3) and is viable (EUROSCARF). med19(rox3) mutants have been shown to be defective for activation of Gal4 (23Brown T.A. Evangelista C. Trumpower B.L. J. Bacteriol. 1995; 177: 6836-6843Crossref PubMed Google Scholar) and Gcn4 (24Qiu H. Hu C. Yoon S. Natarajan K. Swanson M.J. Hinnebusch A.G. Mol. Cell Biol. 2004; 24: 4104-4117Crossref PubMed Scopus (81) Google Scholar)-induced genes. On the other hand, med19(rox3) mutants also lead to derepression of heat shock (16Singh H. Erkine A.M. Kremer S.B. Duttweiler H.M. Davis D.A. Iqbal J. Gross R.R. Gross D.S. Genetics. 2006; 172: 2169-2184Crossref PubMed Scopus (39) Google Scholar), glucose-repressed (18Song W. Treich I. Qian N. Kuchin S. Carlson M. Mol. Cell Biol. 1996; 16: 115-120Crossref PubMed Scopus (110) Google Scholar), and HO genes (19Tabtiang R.K. Herskowitz I. Mol. Cell Biol. 1998; 18: 4707-4718Crossref PubMed Scopus (45) Google Scholar). Gene expression microarray studies of a med19(rox3) deletion strain (25Becerra M. Lombardia-Ferreira L.J. Hauser N.C. Hoheisel J.D. Tizon B. Cerdan M.E. Mol. Microbiol. 2002; 43: 545-555Crossref PubMed Scopus (72) Google Scholar) and a med19(rox3) truncation strain (5van de Peppel J. Kettelarij N. van Bakel H. Kockelkorn T.T. van Leenen D. Holstege F.C. Mol. Cell. 2005; 19: 511-522Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) have shown that the expression of broad sets of genes are both up- and down-regulated. To gain further insight into the unusual functional properties of Med19(Rox3), we purified Mediator complexes from the Δmed19(rox3) strain. Consistent with the Middle module phenotypes caused by med19(rox3) mutants, we found that Med19(Rox3) was critical for the stable association of the Middle module with the complex. In contrast to current models of Mediator structure, we found that release of the Middle module of Mediator left an intact and stable complex between Tail and Head module subunits. Characterization of this complex using binding and transcription assays in vitro has given us further insight into the coordination of Mediator structure and function. Yeast Strains—The Δmed19(rox3) strain was obtained from EUROSCARF (accession number Y03119). To construct the MED18(SRB5)-FLAG-tagged Δmed19(rox3) strain, the NatR marker was swapped for the KanMX4 marker in an otherwise wild type MED18(SRB5)-FLAG strain (SHY349). The strain yLM40 was made by transforming SHY349 (26Rani P.G. Ranish J.A. Hahn S. Mol. Cell Biol. 2004; 24: 1709-1720Crossref PubMed Scopus (42) Google Scholar) with an EcoRI fragment of the plasmid p4339 (27Tong A.H. Boone C. Methods Mol. Biol. 2006; 313: 171-192PubMed Google Scholar) containing the NatR maker and selecting for nourseothricin resistance. A triple FLAG epitope tag was placed at the C terminus of MED18(SRB5) in the Δmed19(rox3) strain, by amplifying the 3′ end of a previously FLAG-tagged copy of MED18(SRB5) from the genomic DNA of the yLM40 strain using the following primers: 5′-GGAGGGTTCCTTTTAAAAGCA-3′ and 5′-GAAGCAAATTGCCAAACA-3′. The PCR product was then used to transform the Δmed19(rox3) strain, and transformants were selected for nourseothricin resistance. The correct integration of the FLAG tag was confirmed by PCR and immunoblotting for FLAG-tagged Med18(Srb5) in the strain yLM45. Protein Purification—The untagged Δmed19(rox3) complexes were prepared by modification of the methods used for conventional purification of the wild type Mediator complex (28Myers L.C. Leuther K. Bushnell D.A. Gustafsson C.M. Kornberg R.D. Methods Comp. Methods Enzymol. 1997; 12: 212-216Crossref Scopus (36) Google Scholar). Specifically, the Δmed19(rox3) strain was grown at 26 °C to an A600 of 2 in YPD (4% glucose). The lysis, as well as the Bio-Rex 70 and DEAE-Sepharose chromatography were carried out as previously described for the wild type complex (28Myers L.C. Leuther K. Bushnell D.A. Gustafsson C.M. Kornberg R.D. Methods Comp. Methods Enzymol. 1997; 12: 212-216Crossref Scopus (36) Google Scholar). The hydroxyapatite column was run as previously described; however, there were two distinct peaks containing Mediator proteins instead of one. The first peak, containing only Middle module subunits, co-eluted at ∼75 mm potassium phosphate, whereas a second peak containing subunits from all three modules co-eluted at ∼125 mm potassium phosphate. The peak containing only Middle module subunits was pooled and applied to a Mono-Q 5/5 column (GE Biosciences) as previously described, except that a 26 column volume linear gradient was run from 200 to 1500 mm potassium acetate. The peak of Middle module subunits co-eluted at ∼925 mm potassium acetate. This peak of Middle module subunits was pooled and applied to a Heparin 5PW column (TosoHass) at 200 mm potassium acetate. The Middle module subunits all flowed through the Heparin column. The flow-through fractions were pooled, concentrated, and used directly for immunoblots. The hydroxyapatite peak containing subunits from all three modules was pooled and applied to a Mono-Q 5/5 column as previously described, except that a 26-column volume linear gradient was run from 200 to 1500 mm potassium acetate. Instead of a single peak of Mediator proteins, a peak of Head and Tail module subunits co-eluted at ∼740 mm potassium acetate, and a separate peak containing Head, Middle, and Tail modules co-eluted at ∼880 mm potassium acetate. The peak of Head and Tail module subunits was pooled and applied to a Heparin 5PW column as described (28Myers L.C. Leuther K. Bushnell D.A. Gustafsson C.M. Kornberg R.D. Methods Comp. Methods Enzymol. 1997; 12: 212-216Crossref Scopus (36) Google Scholar). The Head and Tail module subunits co-eluted from the Heparin column at ∼410 mm potassium acetate. The peak fractions were pooled and used directly for immunoblots. The peak from the Mono-Q column containing subunits from all three modules was pooled and applied to a Heparin 5PW column as described (28Myers L.C. Leuther K. Bushnell D.A. Gustafsson C.M. Kornberg R.D. Methods Comp. Methods Enzymol. 1997; 12: 212-216Crossref Scopus (36) Google Scholar). The Head, Middle, and Tail module subunits co-eluted from the Heparin column at ∼370 mm potassium acetate. The peak fractions were pooled, concentrated, and used directly for immunoblots. Purification of untagged wild type Mediator, as well as the general transcription factors, was carried out as previously described (28Myers L.C. Leuther K. Bushnell D.A. Gustafsson C.M. Kornberg R.D. Methods Comp. Methods Enzymol. 1997; 12: 212-216Crossref Scopus (36) Google Scholar). Affinity Purification of Δmed19(rox3)—The MED18(SRB5)-FLAG-tagged wild type and Δmed19(rox3) strains were grown at 26 °C to an A600 of 2 in YPD (4% glucose) and an extract prepared by the a blender/liquid N2 lysis method (29Takagi Y. Chadick J.Z. Davis J.A. Asturias F.J. J. Biol. Chem. 2005; 280: 31200-31207Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The extract was dialyzed against FLAG buffer (20 mm Hepes-KOH, pH 7.6, 0.01% Nonidet P-40, 10% glycerol, 300 mm KOAc, 1 mm dithiothreitol, and protease inhibitors) and adjusted to a final KOAc concentration of 300 mm by the addition of FLAG buffer with no KOAc. Approximately 20 mg of dialyzed extract was added to 200 μl Anti-FLAG M2 agarose beads (Sigma), incubated for 2h at 4 °C with rotation, and washed four times with 10 ml of FLAG buffer. Mediator was eluted with FLAG buffer containing 100 μg/ml 3× FLAG peptide (Sigma). To evaluate the stability of the wild type and Δmed19(rox3) Mediators, the above protocol was modified to add a wash with 10 ml of FLAG buffer containing 1 m urea subsequent to the 4 × 10-ml washes with FLAG buffer. Before the FLAG peptide elution, a 10-ml wash with FLAG buffer followed the wash with 1 m urea. To obtain a highly purified preparation of the Δmed19(rox3) complex containing only Head and Tail module subunits, a lysate was prepared from the MED18(SRB5)-FLAG-tagged Δmed19(rox3) strain. Starting from this lysate, a complex containing only Head and Tail module proteins was purified as described above for the untagged complex. After the Mono-Q step, the peak fractions containing the Head-Tail (HT) complex were pooled, dialyzed against FLAG-buffer, and adjusted to a final salt concentration of 300 mm KOAc. This sample was than applied to the FLAG-agarose beads, which were washed and eluted as described above. Immunoblot Analyses—Immunoblot analyses with the α-Med2, α-Med4, α-Med7, and α-Med8 antibodies were performed as previously described (7Myers 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 (252) Google Scholar). The α-Med14(Rgr1), α-Med16(Sin4), α-Med9, and α-Med11 antibodies were a gift from Y.-J. Kim. The α-Med18(Srb5) and α-Med20(Srb2) were a gift from R. A. Young. The α-Med15(Gal11) was a gift of T. Fukasawa, and the α-Med1 antibody was a gift of S. Bjorklund. Transcription and Kinase Assays—Basal and activated transcription reconstituted from purified yeast general transcription factors was measured using the G-less cassette assay as previously described (7Myers 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 (252) Google Scholar) with the following modifications. The final salt concentration in the reaction buffer was 165 mm KOAc, and the reactions were preincubated for 10 min in the absence of nucleotides followed by a 60-min reaction time after the addition of the nucleotides. The general transcription factors used for all of the reactions in this study came from identical aliquots of a single preparation. The kinase assays with purified Mediator complexes, TFIIK, and GST-CTD were performed as previously described (30Guidi B.W. Bjornsdottir G. Hopkins D.C. Lacomis L. Erdjument-Bromage H. Tempst P. Myers L.C. J. Biol. Chem. 2004; 279: 29114-29120Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Activator and RNA Pol II Binding Assays—Binding of wild type and Δmed19(rox3)-HT purified Mediator complexes to immobilized GST activator fusion proteins was carried out as previously described (31Hengartner C.J. Thompson C.M. Zhang J. Chao D.M. Liao S.M. Koleske A.J. Okamura S. Young R.A. Genes Dev. 1995; 9: 897-910Crossref PubMed Scopus (189) Google Scholar). To assay the binding of core RNA Pol II to wild type and Δmed19(rox3)-Head-Middle-Tail (HMT) Mediator, a Med18(Srb5)-FLAG-tagged version of each of these complexes were immobilized on FLAG-agarose as described above. The immobilized Mediator beads were equilibrated in FLAG buffer with 200 mm KOAc. Equal amounts of purified core RNA Pol II was added to the wild type and Δmed19(rox3)-HMT Mediator beads. After incubation for 2 h at 4 °C with rotation, the supernatant was collected, and the beads were washed four times with FLAG buffer with 200 mm KOAc. Wild type or Δmed19(rox3)-HMT Mediator and any associated RNA Pol II was eluted with FLAG buffer containing 100 μg/ml 3× FLAG peptide (Sigma). Deletion of Med19(Rox3) Leads to Dissociation of the Middle Module of Mediator, Leaving an Intact Mediator Subcomplex Consisting of Head and Tail Modules—We undertook a conventional purification of complexes containing Mediator subunits from a Δmed19(rox3) strain to determine the structural impact of deleting the Med19(Rox3) subunit from Mediator. We followed the purification by immunoblotting with antibodies against subunits in all three modules of core Mediator. From the Δmed19(rox3) lysate through the first two ion exchange columns (Bio-Rex70 and DEAE-Sepharose), the strain showed an identical fractionation pattern to wild type (Fig. 1A). Fractionation of the DEAE pool containing Δmed19(rox3) Mediator on hydroxyapatite, however, showed a pattern distinct from that of wild type. Instead of a single peak, we observed an initial peak of Middle module subunits, followed by a second peak containing subunits from all three modules (Fig. 1B). To further characterize the peak containing only Middle module subunits, we pooled these fractions and observed that the Middle module subunits co-elute over a Mono-Q column and all flow through a Heparin column (Fig. 1A). The flow through fractions were pooled, concentrated, and compared with wild type Mediator by immunoblotting. This Middle module complex (Δmed19 (rox3)-M) contained stoichiometric amounts of Middle module subunits (compared with wild type) and levels of Head and Tail module subunits that were below detection (Fig. 1D). The second peak on the hydroxyapatite, containing subunits from all three modules, was pooled and further fractionated on a Mono-Q column (Fig. 1A). In contrast to wild type Mediator, two distinct peaks of Mediator eluted from the Mono-Q column. The first peak contained subunits from only the Head and Tail modules of the complex, whereas the second peak contained subunits from all three modules (Fig. 1C). Both of these peaks were individually pooled and applied to a Heparin column. Fractionation of the first pool (Δmed19(rox3)-HT) on Heparin resulted in a peak of Head and Tail module subunits co-eluting from the column (Fig. 1A), whereas fractionation of the second pool (Δmed19(rox3)-HMT) on Heparin resulted in a peak of subunits from all three modules co-eluting from the column (Fig. 1A). These two peaks were individually pooled, concentrated, and compared with wild type Mediator by immunoblotting. The analysis of the (Δmed19(rox3)-HT) complex showed stoichiometric amounts of Head and Tail module subunits (compared with wild type) and levels of Middle module subunits that were below detection (Fig. 1D). The analysis of the (Δmed19(rox3)-HMT) complex showed stoichiometric amounts of subunits from all three modules (compared with wild type) (Fig. 1D). Immunoblotting with an antibody against Med19(Rox3) confirmed that this subunit was absent from all Mediator preparations from the mutant strain (data not shown). Three distinct Δmed19(rox3) Mediator complexes exist after purification. The first is a largely intact complex (Δmed19(rox3)-HMT), missing only the deleted subunit. The second and third (Δmed19(rox3)-M and Δmed19(rox3)-HT) appear to result from the dissociation of the Middle module complex from the Δmed19(rox3)-HMT Mediator. The existence of a stable Δmed19(rox3)-HT complex was surprising; to further characterize the composition of the complex, we took an affinity purification approach. To purify the Δmed19(rox3)-HT complex to homogeneity, we grew cells from a Med18(Srb5)-FLAG-tagged wild type and Δmed19(rox3) strain and followed the conventional purification protocol, as described above, through the Mono-Q stage. After separation on Mono-Q, the fractions containing the Δmed19(rox3)-HT complex were pooled and subjected to a final purification step on FLAG-agarose. Immunoblotting of the pure Δmed19(rox3)-HT complex shows that an affinity tag on the Head module could pull down stoichiometric amounts of Tail module proteins as compared with the wild type complex (Fig. 2A). Immunoprecipitation of Head subunits by an antibody against a Tail subunit (α-Med2) confirmed this result (data not shown). A silver stain analysis of purified Δmed19(rox3)-HT complex shows that the complex appears to contain only Head and Tail module proteins in stoichiometric quantities (Fig. 2C). This result suggests that it is unlikely that additional "non-Mediator" proteins had associated with the complex and "compensated" for the absence of the Middle module subunits. We cannot, however, rule this possibility out given the differential staining of proteins by silver. Contrary to current models, the Head and Tail modules of Mediator can directly form a stable complex in the absence of a Middle module. To determine the composition of the Δmed19(rox3) Mediator under mild conditions, we purified the complex directly from lysate of the MED18(SRB5)-FLAG-tagged Δmed19(rox3) strain on FLAG-agarose. During the conventional purification of the Δmed19(rox3) Mediator, the complex is subjected to several highly stringent conditions that could lead to dissociation of weakly interacting subunits or modules. Immunoblotting of Mediator subunits from affinity-purified Med18(Srb5)-FLAG-tagged wild type and Δmed19(rox3) complexes show the presence of stoichiometric amount of subunits from all three modules, as compared with conventionally purified wild type Mediator (Fig. 2B). Hence, it appears as if the largely intact Δmed19(rox3)-HMT Mediator is the predominant form of the complex under mild conditions. This procedure was an effective means to purify the Δmed19(rox3)-HMT complex to homogeneity. Silver stain analysis confirmed the purity and stoichiometry of the Δmed19(rox3)-HMT Mediator complex purified under mild conditions (Fig. 2C). Absence of Med19(Rox3) Destabilizes Attachment of Middle Module in Intact Mediator Complex—Because a majority of the Δmed19(rox3) Mediator under mild conditions appears to be present in a largely intact form, we sought to determine the origin of the Δmed19(rox3)-HT complex. We hypothesized that the absence Med19(Rox3) leads to the destabilization of the intact complex and that a stringent step during the conventional purification resulted in the partial displacement of Middle module from the complex. To test this idea we immobilized both Med18(Srb5)-FLAG-tagged wild type and Δmed19(rox3)-HMT Mediator on FLAG-agarose and washed the complex with a buffer containing 1 m urea. Earlier studies have shown that the an immobilized wild type Mediator is resistant to 1 m urea but starts to dissociate at concentrations greater than 2 m (32Kang J.S. Kim S.H. Hwang M.S. Han S.J. Lee Y.C. Kim Y.J. J. Biol. Chem. 2001; 276: 42003-42010Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). After treatment with 1 m urea, the Med18(Srb5)-FLAG-tagged wild type and Δmed19(rox3)-HMT Mediator were eluted with FLAG peptide and subjected to immunoblotting. As shown previously, the wild type Mediator rem
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