Preponderance of Free Mediator in the Yeast Saccharomyces cerevisiae
2005; Elsevier BV; Volume: 280; Issue: 35 Linguagem: Inglês
10.1074/jbc.c500150200
ISSN1083-351X
AutoresYuichiro Takagi, James Z. Chadick, Joshua A. Davis, Francisco J. Asturias,
Tópico(s)RNA Research and Splicing
ResumoBiochemical evidence suggesting that the predominant form of Mediator in the yeast Saccharomyces cerevisiae might be one in which the complex is associated with RNA polymerase II to form a holoenzyme has led to the proposition of a holoenzyme-based model for transcription initiation. We report that polymerase-free Mediator, isolated early on during a whole-cell extract fractionation protocol, is in fact the most abundant form of the Mediator complex. The existence of free Mediator would make possible independent recruitment of Mediator and RNA polymerase II to the preinitiation complex. This is in agreement with reports from in vivo studies of time and spatial independence of Mediator and RNA polymerase II promoter interaction, with current models of preinitiation complex structure in which promoter DNA upstream of the transcription start site is positioned between Mediator and polymerase, and with the proposed role of Mediator as the major component of the Scaffold complex involved in transcription reinitiation. Biochemical evidence suggesting that the predominant form of Mediator in the yeast Saccharomyces cerevisiae might be one in which the complex is associated with RNA polymerase II to form a holoenzyme has led to the proposition of a holoenzyme-based model for transcription initiation. We report that polymerase-free Mediator, isolated early on during a whole-cell extract fractionation protocol, is in fact the most abundant form of the Mediator complex. The existence of free Mediator would make possible independent recruitment of Mediator and RNA polymerase II to the preinitiation complex. This is in agreement with reports from in vivo studies of time and spatial independence of Mediator and RNA polymerase II promoter interaction, with current models of preinitiation complex structure in which promoter DNA upstream of the transcription start site is positioned between Mediator and polymerase, and with the proposed role of Mediator as the major component of the Scaffold complex involved in transcription reinitiation. Mediator is a global regulator of transcription, first identified in the yeast Saccharomyces cerevisiae (1Flanagan P.M. Kelleher R. J. III Sayre M.H. Tschochner H. Kornberg R.D. Nature. 1991; 350: 436-438Crossref PubMed Scopus (257) Google Scholar, 2Kelleher I. R. J. Flanagan P.M. Kornberg R.D. Cell. 1990; 61: 1209-1215Abstract Full Text PDF PubMed Scopus (285) Google Scholar), which acts as an interface between gene-specific regulator proteins and the general transcription machinery (3Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (879) Google Scholar, 4Myers 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 (251) Google Scholar). Mediator complexes have been identified in all eukaryotic organisms examined on the basis of moderate sequence homology of their component subunits to corresponding subunits in the yeast complex (5Boube M. Joulia L. Cribbs D.L. Bourbon H.M. Cell. 2002; 110: 143-151Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar) and appear to play a universal role in integrating information for control of gene expression. The limited sequence homology among Mediator subunits of different eukaryotes points to a high degree of evolutionary divergence. However, it has been suggested that sequence conservation could be highest in protein segments involved in inter-subunit interactions, suggesting that the overall organization of Mediator, and perhaps its mechanism, could be conserved (6Guglielmi 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 (173) Google Scholar). In agreement with this scenario, structural studies have revealed a limited degree of structural similarities among Mediator complexes from yeast to man (7Asturias F.J. Jiang Y.W. Myers L.C. Gustafsson C.M. Kornberg R.D. Science. 1999; 283: 985-987Crossref PubMed Scopus (199) Google Scholar, 8Dotson M.R. Yuan C.X. Roeder R.G. Myers L.C. Gustafsson C.M. Jiang Y.W. Li Y. Kornberg R.D. Asturias F.J. Proc. Natl. Acad. Sci. 2000; 97: 14307-14310Crossref PubMed Scopus (152) Google Scholar, 9Chadick J.Z. Asturias F.J. Trends Biochem. Sci. 2005; 30: 264-271Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Mediator was initially identified in a crude yeast fraction as an activity required to relieve inhibition and enable activator response in a partially purified reconstituted RNA polymerase II transcription system (1Flanagan P.M. Kelleher R. J. III Sayre M.H. Tschochner H. Kornberg R.D. Nature. 1991; 350: 436-438Crossref PubMed Scopus (257) Google Scholar, 2Kelleher I. R. J. Flanagan P.M. Kornberg R.D. Cell. 1990; 61: 1209-1215Abstract Full Text PDF PubMed Scopus (285) Google Scholar). The purified activity, dubbed Mediator, was shown to possess three biochemical functions: stimulation of basal transcription, support of activated transcription, and stimulation of CTD 1The abbreviations used are: CTD, C-terminal domain of Rpb1; RNAPII, RNA polymerase II; TFII, general transcription factor for RNA polymerase II; TBP, TATA-binding protein; WCE, yeast whole-cell extract; HA, hemagglutinin; PEI, polyethylenimine; PI, protease inhibitor; HAP, hydroxyapatite resin; EM, electron microscopy; βME, β-mercaptoethanol; TAP, tandem affinity purification; NTA, nitrilotriacetic acid. 1The abbreviations used are: CTD, C-terminal domain of Rpb1; RNAPII, RNA polymerase II; TFII, general transcription factor for RNA polymerase II; TBP, TATA-binding protein; WCE, yeast whole-cell extract; HA, hemagglutinin; PEI, polyethylenimine; PI, protease inhibitor; HAP, hydroxyapatite resin; EM, electron microscopy; βME, β-mercaptoethanol; TAP, tandem affinity purification; NTA, nitrilotriacetic acid. phosphorylation by the kinase activity of TFIIH (3Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (879) Google Scholar). Several Mediator complex subunits are products of genes previously linked to transcription, such as the SRB genes identified by genetic screens for mutations that compensate for CTD truncations (10Nonet M.L. Young R.A. Genetics. 1989; 123: 715-724Crossref PubMed Google Scholar), the SIN4 and RGR1 genes previously identified as participants in activation and repression (11Li Y. Bjorklund S. Jiang Y.W. Kim Y.-J. Lane W.S. Stillman D.J. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10864-10868Crossref PubMed Scopus (216) Google Scholar), and a number of proteins that are products of novel MED genes (4Myers 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 (251) Google Scholar). Research in the last few years has resulted in biochemical characterization of Mediator homologues in higher organisms and identification of their component subunits (5Boube M. Joulia L. Cribbs D.L. Bourbon H.M. Cell. 2002; 110: 143-151Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 6Guglielmi 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 (173) Google Scholar, 12Sato S. Tomomori-Sato C. Parmely T.J. Florens L. Zybailov B. Swanson S.K. Banks C.A. Jin J. Cai Y. Washburn M.P. Conaway J.W. Conaway R.C. Mol. Cell. 2004; 14: 685-691Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). A significant impediment to studying the biochemical properties and structural organization of yeast Mediator stems from the inherent difficulty in obtaining the complex in pure form, free from the other components of the transcription machinery. Although protocols for isolation of free Mediator have been published (4Myers 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 (251) Google Scholar, 13Myers L.C. Leuther K. Bushnell D.A. Gustafsson C.M. Kornberg R.D. Methods. 1997; 12: 212-216Crossref PubMed Scopus (36) Google Scholar), it has proven extremely challenging to obtain reproducible results, 2Y. Tagaki and F. J. Asturias, unpublished results. 2Y. Tagaki and F. J. Asturias, unpublished results. and as a consequence, the bulk of biochemical studies published to date have relied on partially purified holoenzyme (Mediator-RNA polymerase II (RNAPII)) fractions (14Lee Y.C. Min S. Gim B.S. Kim Y.J. Mol. Cell. Biol. 1997; 17: 4622-4632Crossref PubMed Scopus (62) Google Scholar, 15Lee Y.C. Park J.M. Min S. Han S.J. Kim Y. Mol. Cell. Biol. 1999; 19: 2967-2976Crossref PubMed Scopus (132) Google Scholar, 16Myers L.C. Gustafsson C.M. Hayashibara K.C. 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Observation of these large complexes led to the proposition of a “holoenzyme-based” model for transcription initiation in which one large complex containing all required components of the machinery would be recruited to the promoter by an activator. Although the holoenzyme-based model for initiation is largely based on indirect evidence from studies in yeast, it has influenced the interpretation of observations in other systems, with evidence for recruitment of an individual factor interpreted as indication of recruitment of an entire holoenzyme complex (17Gaudreau L. Adam M. Ptashne M. Mol. Cell. 1998; 1: 913-916Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 26Farrell S. Simkovich N. Wu Y. Barberis A. Ptashne M. Genes Dev. 1996; 10: 2359-2367Crossref PubMed Scopus (109) Google Scholar, 27Gaudreau L. Schmid A. Blaschke D. Ptashne M. Horz W. 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To address the discrepancies between the holoenzyme and sequential recruitment models, here we have sought to establish a reliable purification protocol for free Mediator by asking whether free Mediator can be identified and purified from the yeast S. cerevisiae. An improved cell lysis procedure and careful reevaluation of the Mediator purification protocol, made possible by detection and tracking of Mediator in crude whole-cell extracts, revealed that free Mediator can be identified and isolated to near homogeneity and also that free Mediator, not holoenzyme, is the predominant form of Mediator. Consistent with the results from the in vivo chromatin immunoprecipitation analysis and with the isolation of free Mediator in mammalian cells, these data support a model for initiation in which different components of the transcription machinery are sequentially recruited and Mediator functions as a separate entity. In this scenario, the Mediator-RNAPII holoenzyme is a transient complex formed only during the initiation process. Construction of Yeast-tagging Vector—The epitope-tagging vector, pYT006, was created by modifying the vector pU6H3HA (36De Antoni A. Gallwitz D. Gene. 2000; 246: 179-185Crossref PubMed Scopus (86) Google Scholar) using a QuikChange kit (Stratagene); the His6 tag was disabled by mutating the first four histidine residues to glycines (yielding the vector pYT005), and the sequence of the PreScission protease site (LEVLFQGP) was introduced upstream of three copies of the influenza HA epitope (yielding the vector pYT006). Construction of Tagged Yeast Strains—The yeast CEN vector carrying wild-type Med17 pCT127, was recovered from the yeast strain Z579 (37Thompson C.M. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4587-4590Crossref PubMed Scopus (208) Google Scholar). A His10 tag was introduced into the N terminus of the Med17 open reading frame using a QuikChange kit (Strategene), yielding the plasmid pCT127 (His10-Med17). Three copies of the HA epitope were introduced into the C terminus of the Med8 subunit using the PCR product of pYT006 as a template, with primer sets targeting Med8 genomic locus as described (36De Antoni A. Gallwitz D. Gene. 2000; 246: 179-185Crossref PubMed Scopus (86) Google Scholar). The PCR product was used to transform the yeast strain Z572 (MATa his3Δ200 leu2-3, 112 ura3-52 med17Δ2::HIS3 (CEN, URA3, MED17), Med8::Med8-PreScission-3xHA-Kan) yielding strain YT108. The plasmid, pCT127 (His10-Med17) was then transformed into strain YT108 by plasmid shuffling, yielding strain YT110 (His10-Med17, Med8-PreSci-3xHA). Analysis of Yeast Genomic DNA—Approximately 50 μl of each sample was mixed with 150 μl of buffer containing 50 mm Tris-HCl (pH 8.0) and 20 mm EDTA and incubated with 120 μg of RNase A for 30 min at 37 °C. 10 μl of 10% SDS and 8 units of protease K (Sigma-Aldrich) were added and incubated for an additional 30 min at 37 °C followed by phenol/chloroform extraction and ethanol precipitation. 20 μg of glycogen was then added as a carrier and one-fifth of the extracted DNA was subjected to 1% agarose gel electrophoresis and detected by ethidium bromide staining. Purification of Free Mediator—2 kg of yeast strain YT110 was grown in 2× YPD medium as described (38Takagi Y. Komori H. Chang W.H. Hudmon A. Erdjument-Bromage H. Tempst P. Kornberg R.D. J. Biol. Chem. 2003; 278: 43897-43900Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) to an OD600 of 8.0 and harvested at 3000 × g for 10 min. Cells were washed in ddH2O and frozen at -80 °C. To lyse yeast cells, 220 g of frozen cells, 100 g of dry ice, and ∼300 ml of liquid nitrogen were placed in a 2-liter stainless steel blender jar. This mixture was blended at high speed for 20 min while maintaining the level of liquid nitrogen required to allow the mixture to flow during blending. The whole-cell extract was prepared as described previously (38Takagi Y. Komori H. Chang W.H. Hudmon A. Erdjument-Bromage H. Tempst P. Kornberg R.D. J. Biol. Chem. 2003; 278: 43897-43900Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), except that 600 mm KOAc was used for extraction. The whole-cell extract (WCE) was dialyzed against buffer A (50 mm HEPES-KOH (pH 7.6), 1 mm EDTA, 10% glycerol, 5 mm β-mercaptoethanol (βME), 0.5× protease inhibitor (PI) mix (100× PI mix contains 0.6 mm leupeptin, 2 mm pepstatin A, 2 mm benzamidine, and 1 mm phenylmethylsulfonyl fluoride) and adjusted to the conductivity of buffer A containing 100 mm KOAc (A100). The sample was applied to a 2.5-liter Bio-Rex70 (Bio-Rad) column equilibrated with buffer A100 at 1 column volume/3 h. The resin was then washed with 2 column volumes of buffer A100 followed by 2 column volumes of buffer A400, and the sample was eluted using 2 column volumes of buffer A650. Fractions containing both Med17 and Med8 were pooled, dialyzed against buffer B (50 mm Tris acetate (pH 7.6), 0.1 mm EDTA, 0.01% Nonidet P-40, 10% glycerol, 5 mm βME, 0.5× PI mix) and adjusted to the conductivity of buffer B containing 100 mm KOAc (B100). The sample was applied to a 220-ml DEAE-Sephacel (Sigma-Aldrich) column pre-equilibrated with buffer B100 at 1 column volume/h. The column was washed with 1 column volume of buffer B100 and eluted with a linear gradient from 100 to 550 mm KOAc over 10 column volumes, with the peak Mediator fractions eluting at ∼410 mm KOAc. Fractions containing both Med17 and Med8 but not containing the CTD region of RNAPII were pooled, and CaCl2 was added to a final concentration of 0.2 mm. The sample was applied to a 80-ml hydroxyapatite (Bio-Rad) column pre-equilibrated with buffer H10 (10 mm potassium phosphate (pH 7.8), 100 mm KOAc, 50 μm CaCl2, 10% glycerol, 0.01% Nonidet P-40, 5 mm βME, 0.5× PI mix) at 1 column volume/h. The column was then washed with 1 column volume of buffer H10 and eluted with a linear gradient from 10 to 200 mm potassium phosphate over 10 column volumes. Peak Mediator fractions appeared at ∼110 mm potassium phosphate. Mediator-containing fractions were pooled and dialyzed against buffer Q (50 mm Tris acetate (pH 7.6), 10% glycerol, 5 mm βME, 0.5× PI mix) and adjusted to the conductivity of buffer Q containing 100 mm KOAc (Q100). The sample was applied to a UnoQ6 column (Bio-Rad) pre-equilibrated with buffer Q100 at 1.5 ml/min, and the column was washed with 1 column volume of buffer Q100 followed by 5 column volumes of buffer Q400 and eluted with a linear gradient from 400 to 1200 mm KOAc over 10 column volumes. Peak Mediator fractions appeared at 550 mm KOAc. Mediator fractions were pooled and dialyzed against buffer N (50 mm HEPES-KOH (pH 8.5), 1000 mm KOAc, 0.01% Nonidet P-40, 10% glycerol, 5 mm βME, 0.5× PI mix) containing 10 mm imidazole (N10). Sample was applied to 5 ml of Ni2+ resin (HIS-Select, Sigma-Aldrich) pre-equilibrated with buffer N10, and the resin/sample mixture was continuously agitated at 4 °C for 18 h. The resin was allowed to drain and was then washed with 50 ml of buffer N20 over a period of 30 min and eluted with 10 ml of buffer N300 using 1 ml aliquots over a period of 45 min. Sample was dialyzed against buffer S (50 mm HEPES-KOH (pH 7.6), 5% glycerol, 5 mm βME, 0.5× PI mix) and adjusted to the conductivity of buffer S containing 100 mm KOAc (S100). Sample was applied to a 0.16-ml UNO-S column (Bio-Rad) pre-equilibrated with buffer S100 at 0.35 ml/min and eluted using 5 ml of buffer S700. Production of Antibodies—Polyclonal antibodies against the Med17 subunit were generated (Covance, PA) by inoculating rabbits with the peptide DNDKNLKFLKNKDSLV (Med17 amino acids 72-87) conjugated with KLH. Polyclonal antibodies against the HA epitope were generated (at The Scripps Research Institute) by inoculating rabbits with two copies of the HA peptides, CPDYAGYPYDVPDYAGYPYDV, conjugated with KLH. Antibodies were affinity-purified by glutathione S-transferase-HA column as described (39Kellogg D.R. Moazed D. Methods Enzymol. 2002; 351: 172-183Crossref PubMed Scopus (10) Google Scholar). Production of polyclonal antibody against the Med18 subunit is described elsewhere. 3Y. Takagi and R. D. Kornberg, submitted for publication. Western Blot Analysis—5-μl samples were subjected to 4-20% gradient SDS-PAGE (Bio-Rad). Western blot analysis was carried out as described (38Takagi Y. Komori H. Chang W.H. Hudmon A. Erdjument-Bromage H. Tempst P. Kornberg R.D. J. Biol. Chem. 2003; 278: 43897-43900Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and probed with anti-CTD (8WG16 monoclonal, Abcam, Cambridge MA), anti-Med17, anti-Med18, and anti-HA antibodies. Small Scale Immunoaffinity Purification of Mediator and Stability of the RNAPII-Mediator Holoenzyme—A small amount of whole-cell extract was prepared as described above but using the following low-salt extraction method. 50 g of yeast cells (from YT110 strain) were lysed using the blender method and extracted with 50 ml of 2× low-salt lysis buffer (100 mm HEPES-KOH (pH 7.6), 400 mm KOAc, 2 mm EDTA, 20% glycerol, 0.01% Nonidet P-40, 10 mm βME, and 2× PI mix). The salt concentration of the lysis buffer was carefully adjusted so that the final conductivity of the WCE was close to that of buffer A300. Half of the WCE obtained was loaded onto a 0.6 ml anti-HA antibody column (Sigma-Aldrich) pre-equilibrated with buffer A300 and incubated for 4 h at 4 °C. The resin was washed with buffer A300 until no protein could be detected in the washes and then divided into two 0.3-ml columns. One of the 0.3-ml resin fractions was washed again with buffer A300. The other 0.3-ml resin fraction was washed with a total of 5 column volumes of buffer A600, and 5 fractions were collected. The resin was further washed with a total of 5 column volumes of buffer A1000, and 5 more fractions were collected. Finally, the resin fraction subjected to high-salt washes was equilibrated with buffer A300. Elutions from both low- and high-salt washed resin fractions were carried out by incubation with elution buffer containing 0.5 mg/ml 2× HA peptide for 15 min at 30 °C. Fractions from the 600 and 1000 mm washes, as well as peak elution fractions, were analyzed by Western blot using anti-CTD, anti-Med17, and anti-Med18 antibodies. Both sets of elutions were further analyzed by quantitative Western blot to estimate the amount of RNAPII and Mediator, using purified RNAPII and recombinant Med17 and Med18 proteins 3Y. Takagi and R. D. Kornberg, submitted for publication. as standards. From the quantitative Western blot results, the free Mediator to holoenzyme molar ratio was estimated by assuming that the signal from RNAPII represents holoenzyme and that the Mediator signal arises from the combined signal from free Mediator and holoenzyme. Specific Transcription and CTD Phosphorylation Assays—Reconstituted transcription was performed essentially as described (3Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (879) Google Scholar) with minor modifications. UnoS fractions containing purified free Mediator were dialyzed against a buffer containing 50 mm HEPES-KOH (pH 7.6), 150 mm KOAc, 20% glycerol, and 5 mm βME for 1 h at 4 °C. All factors (RNAPII, TFIIF, TFIIB, TBP, TFIIE, TFIIH, and for some experiments, Mediator and/or GCN4) and the DNA template were mixed and preincubated for 5 min. Transcription was initiated by the addition of a nucleotide mix containing ATP, CTP, and [γ-32P]UTP. This reaction mixture was incubated for an additional 45 min at 24 °C, and the final concentration of cold UTP was adjusted to 10 μm. Transcripts were resolved by denaturing gel electrophoresis followed by autoradiography. The CTD phosphorylation assay was carried out as described previously (3Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (879) Google Scholar) except that 4-15% SDS-PAGE was used to resolve the phosphorylated Rpb1 subunit. Electron Microscopy Analysis—Samples were diluted to 35 μg/ml with 50 mm HEPES-KOH (pH 7.6), and 3.2-μl samples were applied to carbon-coated 400-mesh copper and rhodium grids (Ted Pella, Redding, CA) that were glow-discharged in the presence of amyl amine for 1 min. After 1 min of absorption, the samples were blotted dry and washed three times with a 1% solution of uranyl acetate. The sample was then immersed in 1% uranyl acetate, a second layer of carbon was applied to the top (40Tischendorf G.W. Zeichhardt H. Stoffler G. Mol. Gen. Genet. 1974; 134: 187-208Crossref PubMed Scopus (108) Google Scholar, 41Stoffler G. Stoffler-Meilicke M. Tesche H. Modern Methods in Protein Chemistry. De Gruyter, Berlin1983: 409-455Crossref Google Scholar), and the samples were dried. Samples were imaged using a Phillips CM120 transmission electron microscope outfitted with a LaB6 filament and operated at 100 kV. Images were recorded on Kodak SO-163 film at ×60,000 magnification and digitized using a 7 μm step size on a Zeiss SCAI scanner. Mass Spectroscopy Analysis—5 μg of protein in 25 μl of buffer S700 was precipitated by adding an equal volume of 20% trichloroacetic acid and incubated for 30 min on ice. Samples were pelleted by spinning at 13,000 × g for 10 min and washed three times in ice-cold acetone. The washed pellets were resuspended in 100 mm Tris-HCl (pH 8.5), denatured with 8 m urea, and reduced and alkylated by incubation with 5 mm tris(2-carboxyethyl)phosphine for 30 min at room temperature followed by incubation with 10 mm iodoacetamide for 30 min at room temperature. Samples were divided into three aliquots and subjected to three separate digests. For trypsination, the urea concentration was adjusted to 2 m using 100 mm Tris-HCl (pH 8.5), trypsin (10 ng/μl) was added in the presence of 2 mm CaCl2, and the mixture was agitated overnight at 37 °C. For elastase treatment, the urea concentration was reduced to 2 m as above, elastase was added to a final concentration of 5 ng/μl, and the sample was incubated overnight at 37 °C. For subtilysin digests, urea concentration was reduced to 4 m, and 10 ng of subtilysin was added. The reaction was allowed to proceed for 1 h at 37 °C. Samples were loaded on a 4-cm, 250-micron internal diameter column packed with 5 μm reverse-phase beads coupled to a 3-cm column with a strong cation-exchange resin and a final 10-cm section of reverse-phase media. Peptides were eluted directly to the electrospray ionization tandem mass spectrometer using a gradient of 100 to 0% acetonitrile followed by stepwise elution using 10, 25, 35, 50, 65, 80, and 100% 500 mm ammonium acetate. Electrospray ionization was done at 2.5 kV, and a Thermo Finnigan LTQ was used to acquire data during the entire elution procedure. The nine largest spectral peaks were searched against the S. cerevisiae genome data base using the SEQUEST program and were sorted and filtered using the DTASelect program for each protein sequence containing at least three identified peptide fragments (42MacCoss M.J. Wu C.C. Yates III, J.R. Anal. Chem. 2002; 74: 5593-5599Crossref PubMed Scopus (339) Google Scholar, 43Tabb D.L. McDonald W.H. Yates III, J.R. J. Proteome Res. 2002; 1: 21-26Crossref PubMed Scopus (1113) Google Scholar). Monitoring Mediator in WCE and Optimization of WCE Preparation—Attempts to implement a Mediator immunoaffinity purification protocol from a crude whole-cell extract throu
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