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• W1963940982 abstract "The fundamental transcription initiation factor (TIF) for ribosomal RNA expression by eukaryotic RNA polymerase I, TIF-IB, has been purified to near homogeneity fromAcanthamoeba castellanii using standard techniques. The purified factor consists of the TATA-binding protein and four TATA-binding protein-associated factors with relative molecular weights of 145,000, 99,000, 96,000, and 91,000. This yields a calculated native molecular weight of 460,000, which compares well with its mass determined by scanning transmission electron microscopy (493,000) and its sedimentation rate, which is close to RNA polymerase I (515,000). Both impure and nearly homogeneous TIF-IB exhibit an apparent equilibrium dissociation constant of 56 ± 3 pm. However, although impure TIF-IB can form a promoter-DNA complex resistant to challenge by other promoter-containing DNAs, near homogeneous TIF-IB cannot do so. An additional transcription factor, dubbed TIF-IE, restores the ability of near homogeneous TIF-IB to sequester DNA into a committed complex. The fundamental transcription initiation factor (TIF) for ribosomal RNA expression by eukaryotic RNA polymerase I, TIF-IB, has been purified to near homogeneity fromAcanthamoeba castellanii using standard techniques. The purified factor consists of the TATA-binding protein and four TATA-binding protein-associated factors with relative molecular weights of 145,000, 99,000, 96,000, and 91,000. This yields a calculated native molecular weight of 460,000, which compares well with its mass determined by scanning transmission electron microscopy (493,000) and its sedimentation rate, which is close to RNA polymerase I (515,000). Both impure and nearly homogeneous TIF-IB exhibit an apparent equilibrium dissociation constant of 56 ± 3 pm. However, although impure TIF-IB can form a promoter-DNA complex resistant to challenge by other promoter-containing DNAs, near homogeneous TIF-IB cannot do so. An additional transcription factor, dubbed TIF-IE, restores the ability of near homogeneous TIF-IB to sequester DNA into a committed complex. transcription initiation factor TATA-binding protein TBP-associated factor TAF associated with RNA polymerase I core factor upstream binding factor upstream activation factor electrophoretic mobility shift assay. In eukaryotic transcription, a fundamental transcription initiation factor marks the promoter for subsequent events leading to the recruitment of RNA polymerase (1Paule M.R. Nature. 1990; 344: 819-820Crossref PubMed Scopus (11) Google Scholar). TFIIIB serves this role for RNA polymerase III transcription (2Kassavetis G.A. Braun B.R. Nguyen L.H. Geiduschek E.P. Cell. 1990; 60: 235-245Abstract Full Text PDF PubMed Scopus (360) Google Scholar), TFIID for RNA polymerase II (3Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (628) Google Scholar), and for ribosomal RNA genes, this fundamental factor is dubbed TIF-IB,1 SL1, factor D, Rib1, core factor, or TFID (reviewed in Refs. 1Paule M.R. Nature. 1990; 344: 819-820Crossref PubMed Scopus (11) Google Scholar and 4Paule M.R. Transcription of Eukaryotic Ribosomal RNA Genes by RNA Polymerase I. Springer-Verlag New York Inc., New York1998Google Scholar, 5Paule M.R. Conaway R. Conaway J. Transcription: Mechanism and Regulation. Raven Press, New York1994: 83-106Google Scholar, 6Grummt I. Paule M.R. Transcription of Eukaryotic Ribosomal RNA Genes by RNA Polymerase I. Springer-Verlag New York Inc., New York1998: 135-154Google Scholar, 7Nomura M. Paule M.R. Transcription of Eukaryotic Ribosomal RNA Genes by RNA Polymerase I. Springer-Verlag New York Inc., New York1998: 155-172Google Scholar, 8Zomerdijk J.C.B.M. Tjian R. Paule M.R. Transcription of Eukaryotic Ribosomal RNA Genes by RNA Polymerase I. Springer-Verlag New York Inc., New York1998: 67-94Google Scholar). These factors all contain a common subunit, the TATA-binding protein (TBP) (9Comai L. Tanese N. Tjian R. Cell. 1992; 68: 965-976Abstract Full Text PDF PubMed Scopus (309) Google Scholar, 10Hernandez N. Genes Dev. 1993; 7: 1291-1308Crossref PubMed Scopus (564) Google Scholar), which is associated with a variable number of polymerase-specific subunits, the TATA-binding protein associated factors (TAFs). In human SL1, TBP is associated with three TAFs with M r = 110,000, 63,000, and 48,000 (9Comai L. Tanese N. Tjian R. Cell. 1992; 68: 965-976Abstract Full Text PDF PubMed Scopus (309) Google Scholar); in mouse, the TAFIs haveM r = 95,000, 68,000, and 48,000 (11Eberhard D. Tora L. Egly J.-M. Grummt I. Nucleic Acids Res. 1993; 21: 4180-4186Crossref PubMed Scopus (75) Google Scholar). Lower eukaryotes contain a slightly modified factor. InSaccharomyces cerevisiae, a genetically identified complex called core factor or CF, made up of Rrn6p, Rrn7p, and Rrn11p, appears to function on the core promoter in a manner similar to vertebrate TIF-IB/SL1 (12Keys D.A. Vu L. Steffan J.S. Dodd J.A. Yamamoto R.T. Nogi Y. Nomura M. Genes Dev. 1994; 8: 2349-2362Crossref PubMed Scopus (78) Google Scholar, 13Lalo D. Steffan J.S. Dodd J.A. Nomura M. J. Biol. Chem. 1996; 271: 21062-21067Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 14Lin C.W. Moorefield B. Pavel J.P. Aprikian P. Mitomo K. Reeder R.H. Mol. Cell. Biol. 1996; 16: 6436-6443Crossref PubMed Scopus (60) Google Scholar). Surprisingly, none of the cloned Rrn6/7 or -11 genes from the S. cerevisiae complex show any sequence similarity to the vertebrate TAFIs, and TBP is not tightly associated with the TAFIs. In Acanthamoeba castellanii, a single transcription factor, TIF-IB, is necessary and sufficient to form a complex on the promoter and recruit RNA polymerase I for multiple rounds of transcription (15Bateman E. Iida C.T. Kownin P. Paule M.R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8004-8008Crossref PubMed Scopus (38) Google Scholar, 16Iida C.T. Kownin P. Paule M.R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1668-1672Crossref PubMed Scopus (40) Google Scholar, 17Kownin P. Bateman E. Paule M.R. Cell. 1987; 50: 693-699Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 18Bateman E. Hoffman L. Iida C. Kubaska W. Kownin P. Risi P. Zwick M. Paule M.R. Cech T. Gralla J. Molecular Biology of RNA and DNA-Protein Interactions in Transcription. Alan R. Liss, Inc., New York1989: 259-269Google Scholar, 19Paule M.R. Paule M.R. Transcription of Eukaryotic Ribosomal RNA Genes by RNA Polymerase I. Springer-Verlag New York Inc., New York1998: 107-120Google Scholar). The ability of TIF-IB to form a stable complex on the core promoter varies considerably from species to species. At one end of the spectrum, human SL1 can bind only very weakly or not at all to the rRNA promoter by itself, based on its ability to mediate transcriptionin vitro in the presence of only RNA polymerase I (20Zomerdijk J.C.B.M. Tjian R. Paule M.R. Transcription of Eukaryotic Ribosomal RNA Genes by RNA Polymerase I. Springer-Verlag New York Inc., New York1998: 121-134Google Scholar). Instead, an accessory factor, upstream binding factor (UBF), binds first to an upstream promoter element and apparently aids the binding of SL1 either by interacting with it (21Bell S.P. Learned R.M. Jantzen H.-M. Tjian R. Science. 1988; 241: 1192-1197Crossref PubMed Scopus (263) Google Scholar) or by altering the structure of the DNA in the region bound by SL1 (22Bazett-Jones D.P. Leblanc B. Herfort M. Moss T. Science. 1994; 264: 1134-1137Crossref PubMed Scopus (204) Google Scholar). The UBF footprint persists following committed complex formation, showing it remains in the complex (21Bell S.P. Learned R.M. Jantzen H.-M. Tjian R. Science. 1988; 241: 1192-1197Crossref PubMed Scopus (263) Google Scholar). In Xenopus laevis, Rib1 similarly cannot form a stable complex without UBF, but for a different reason; in addition to its ability to alter the DNA structure of the promoter (22Bazett-Jones D.P. Leblanc B. Herfort M. Moss T. Science. 1994; 264: 1134-1137Crossref PubMed Scopus (204) Google Scholar), in a DNA-independent mechanism UBF prevents the dissociation of TBP from the rather unstable Rib1 (23Bodeker M. Cairns C. McStay B. Mol. Cell. Biol. 1996; 16: 5572-5578Crossref PubMed Scopus (13) Google Scholar). In rat and mouse, UBF can also stimulate TIF-IB binding. However, in these species UBF is not required for stable association of the TBP-containing factor with the promoter, and thus TIF-IB is sufficient for specific initiation in these systems (6Grummt I. Paule M.R. Transcription of Eukaryotic Ribosomal RNA Genes by RNA Polymerase I. Springer-Verlag New York Inc., New York1998: 135-154Google Scholar,24Smith S.D. O'Mahony D.J. Kinsella B.T. Rothblum L.I. Gene Expr. 1993; 3: 229-236PubMed Google Scholar). A. castellanii TIF-IB is at the farthest extreme of the spectrum of TBP·TAFI complexes in its ability to interact very strongly with the promoter in the absence of any other assembly or architectural proteins. Recently, a distinct transcription factor dubbed upstream activation factor (UAF) has been genetically identified in Saccharomyces cerevisiae. Like UBF, this multiprotein complex functions when bound to the upstream promoter element, stabilizing binding of CF to the DNA (25Keys D.A. Lee B.S. Dodd J.A. Nguyen T.T. Vu L. Fantino E. Burson L.M. Nogi Y. Nomura M. Genes Dev. 1996; 10: 887-903Crossref PubMed Scopus (118) Google Scholar). However, UAF can commit the template, but CF cannot. Curiously, UAF appears to act on CF via a bridging TBP molecule. The latter is reminiscent of one of the roles of Xenopus UBF, stabilizing the interaction of TBP with the TAFIs (23Bodeker M. Cairns C. McStay B. Mol. Cell. Biol. 1996; 16: 5572-5578Crossref PubMed Scopus (13) Google Scholar). Whether UBF or UAF homologs are obligatory parts of the committed complex in A. castellanii is not clear. We have shown that the DNA in the A. castellanii committed complex is not wrapped or looped (26Gong X. Radebaugh C.A. Geiss G.K. Simon M.N. Paule M.R. Mol. Cell. Biol. 1995; 15: 4956-4963Crossref PubMed Scopus (18) Google Scholar) as in a UBF complex (22Bazett-Jones D.P. Leblanc B. Herfort M. Moss T. Science. 1994; 264: 1134-1137Crossref PubMed Scopus (204) Google Scholar, 27Putnam C.D. Copenhaver G.P. Denton M.L. Pikaard C.S. Mol. Cell. Biol. 1994; 14: 6476-6488Crossref PubMed Scopus (73) Google Scholar), and we cannot identify an upstream promoter element in vitro or a UBF or UAF footprint consistent with those found in vertebrates or yeast. TBP is a stable subunit of A. castellanii TIF-IB (28Radebaugh C.A. Matthews J.L. Geiss G.K. Liu F. Wong J.-M. Bateman E. Camier S. Sentenac A. Paule M.R. Mol. Cell. Biol. 1994; 14: 597-605Crossref PubMed Scopus (43) Google Scholar). Thus,A. castellanii TIF-IB appears to have some functions not found in the other factors. In this paper, we describe a procedure to obtain A. castellanii TIF-IB in a nearly homogeneous form. We show that polypeptides with apparent molecular weights of 145,000, 99,000, 96,000, and 91,000 copurify with TBP and TIF-IB activity. The homogeneous TIF-IB was functionally tested. It is capable of driving specific transcription initiation in an in vitro system consisting of only TIF-IB and RNA polymerase I purified to near homogeneity. However, unlike TIF-IB from earlier stages of purification, homogeneous TIF-IB is incapable of forming as persistent a complex with the core promoter in a template commitment assay. Despite this finding, the apparent dissociation constant between TIF-IB and the promoter-DNA is identical between fractions capable and incapable of template commitment. The ability to commit the template can be restored by adding a partially purified factor, which we dub TIF-IE. 2Although there is no TIF-ID in the literature, the TBP·TAFI complex has been named factor D or TFID, so to avoid confusion “TIF-ID” is not used. The standard assay for TIF-IB was carried out in a final volume of 50 μl containing a 500 μm concentration each of ATP, CTP, and UTP; 25 μm GTP; 5 μCi of [α-32P]GTP (NEN Life Science Products; 3000 Ci/mmol); 100 mm KCl; 10 mm MgCl2; 20 mm HEPES-KOH (pH 7.9); 10% (v/v) glycerol; 0.1 mm EDTA; 0.5 mm dithiothreitol; and 50 ng of linearized plasmid DNA, pEBH10/NdeI (15Bateman E. Iida C.T. Kownin P. Paule M.R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8004-8008Crossref PubMed Scopus (38) Google Scholar) or pAr6/HindIII. pAr6, which contains the rRNA promoter from −683 to +219, was derived from pAr4 (29Perna P.J. Harris G.H. Iida C.T. Kownin P. Bugren S. Paule M.R. Gene Expr. 1992; 2: 71-78PubMed Google Scholar) by excision of theEcoRI–DdeI fragment encompassing the transcription initiation site, filling in its ends with the Klenow fragment of DNA polymerase I, and ligating it into the SmaI site of pUC8. The reactions were started by the addition of individual fractions (1–5 μl) containing TIF-IB and 30 milliunits of heparin-Sepharose-purified RNA polymerase I (30Spindler S. Duester G.L. D'Alessio J.M. Paule M.R. J. Biol. Chem. 1978; 253: 4669-4675Abstract Full Text PDF PubMed Google Scholar). Incubation was at 25 °C for 30 min. The reactions were terminated by the addition of 50 μl of stop buffer containing 1 mg/ml proteinase K and 1% SDS, followed by incubation at 50 °C for 60 min. Nucleic acids were precipitated by the addition of 200 μl of 3 m ammonium acetate, 0.125 mg/ml linear polyacrylamide, and 750 μl of 95% ethanol. Nucleic acids were pelleted in an Eppendorf centrifuge for 30 min at maximum speed. The pellets were washed with 1 ml of 75% ethanol; dried under vacuum; suspended in 5 μl of 95% deionized formamide, 10 mm EDTA, 0.1% bromphenol blue, 0.1% xylene cyanol; and analyzed on a denaturing (7 m urea) 6% polyacrylamide sequencing gel, 0.5× Tris borate-EDTA (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 1989; (and B.23, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY): 18.55Google Scholar). TIF-IB activity was determined in a run-off transcription assay by carrying out a titration of each pool containing TIF-IB. The specific radioactivity of GTP in the assay and the number of nmol of incorporated GMP were determined by simultaneous exposure of phosphor storage screens to a known volume of the reaction mixture containing [α-32P]GTP and the runoff products in the dried polyacrylamide gel, followed by quantification using ImageQuant version 4.1 software. One unit of TIF-IB activity is defined as the amount mediating incorporation of 1 nmol of [32P]GMP into a 309-nucleotide run-off in the standard 30 min assay. The 309-nucleotide run-off product contains 76 G residues. The minimum amount of template required to bind all the available TIF-IB in the reaction was determined for each template and used in the following protocol. Template (pAr6 and/or pEBH10) was preincubated with TIF-IB or TIF-IB plus TIF-IE under the standard assay conditions for 10 min, except RNA polymerase I was omitted. Since some of the experiments described here were done with very pure and dilute components, bovine serum albumin (0.5 mg/ml) was included in the preincubation mixtures to stabilize the protein components and help prevent them from binding nonspecifically to the walls of the reaction vessel. The second template or buffer was added, and preincubation continued for another 10 min. RNA polymerase I was added to start the RNA synthesis phase, which proceeded for another 30 min. Run-off RNAs were analyzed as described above. Electrophoretic mobility shift assays were carried out as described (28Radebaugh C.A. Matthews J.L. Geiss G.K. Liu F. Wong J.-M. Bateman E. Camier S. Sentenac A. Paule M.R. Mol. Cell. Biol. 1994; 14: 597-605Crossref PubMed Scopus (43) Google Scholar), except the electrophoretic buffer was the Tris-glycine buffer used for protein gels minus the SDS (32Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor, NY1982: 348Google Scholar), and electrophoresis was carried out at room temperature. An accurately known amount of promoter DNA (EcoRI/HindIII-cut pEBH10; −120 to +80) was labeled with 32P by fill-in with the Klenow fragment of DNA polymerase I, and its specific activity was estimated by spotting on DEAE filter paper discs (DE81, Whatman, Fairfield, NJ) and washing as for an RNA polymerase nonspecific assay (30Spindler S. Duester G.L. D'Alessio J.M. Paule M.R. J. Biol. Chem. 1978; 253: 4669-4675Abstract Full Text PDF PubMed Google Scholar). A fixed amount of TIF-IB was titrated with known amounts of this labeled DNA as described, except no competitor DNA was added (28Radebaugh C.A. Matthews J.L. Geiss G.K. Liu F. Wong J.-M. Bateman E. Camier S. Sentenac A. Paule M.R. Mol. Cell. Biol. 1994; 14: 597-605Crossref PubMed Scopus (43) Google Scholar), and the resulting mixture analyzed in an EMSA as described above. ImageQuant version 4.1 software was used to analyze the amount of free DNA and TIF-IB·DNA complex present at each DNA concentration, which was then plotted according to the method of Scatchard. The kinetic dissociation rate constant for the TIF-IB·DNA complex was estimated by forming the complex for 10 min, as above, and then adding a 100-fold molar excess of unlabeled promoter DNA for various time periods. In the experiment shown in Fig. 7, start times for each time point were staggered so that all the incubations ended simultaneously. At the end of the incubation, the samples were rapidly chilled on ice, immediately loaded onto the EMSA gel, and electrophoresed. Data were analyzed by Phosphor Imager analysis. Proteins were precipitated with chloroform-methanol (33Wessel D. Flugge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3191) Google Scholar), resuspended in 1× SDS loading buffer and electrophoresed through an SDS-10% or 7.5% polyacrylamide gel by standard methods (34Garfin D.E. Deutscher M.P. Guide to Protein Purification. Academic Press, Inc., San Diego1990: 425-441Google Scholar). Gels were stained using Coomassie Brilliant Blue R-250 as described (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 1989; (and B.23, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY): 18.55Google Scholar) or with silver (35Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3742) Google Scholar). RNA polymerase I was purified from a whole cell extract as described by Spindler et al. (30Spindler S. Duester G.L. D'Alessio J.M. Paule M.R. J. Biol. Chem. 1978; 253: 4669-4675Abstract Full Text PDF PubMed Google Scholar). The TIF-IE used in the study described herein was separated from RNA polymerase I at the last step, glycerol gradient rate zonal sedimentation, but the yield is variable from successive preparations of RNA polymerase I. We have recently found a larger pool of TIF-IE in the BioRex70 fraction of TIF-IB, which can be further purified by heparine-Ultrogel A4R (IBF Biotechnics, Paris) and rate-zonal sedimentation (data not shown). In both cases, TIF-IE is not yet homogeneous. Both strands of the A. castellanii rRNA promoter from −70 to −15 were chemically synthesized incorporating five point mutations (G−53A, C−51T, G−37T, C−22T, and G−20C) which increase promoter strength, presumably by increasing the binding strength to TIF-IB (36Kownin P. Bateman E. Paule M.R. Mol. Cell. Biol. 1988; 8: 747-753Crossref PubMed Scopus (22) Google Scholar). Four base 5′-extensions (G residues at −70 and C residues at −15) were included so that the annealed oligonucleotide could be oligomerized. The ends of the oligomerized DNA were filled using the Klenow fragment of Escherichia coli DNA polymerase I and cloned into the HincII site of Bluescribe(−) (Stratagene, La Jolla, CA). A fragment containing four head-to-tail copies of the −70 to −15 promoter sequence were excised using EcoRI and HindIII, purified over Sephacryl S-500, and coupled to cyanogen bromide-activated Sepharose CL-4B as described (37Kadonaga J.T. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5889-5893Crossref PubMed Scopus (718) Google Scholar), except unreacted cyanogen bromide-derivatized Sepharose was inactivated with 1m Tris (pH 8). Protein concentration in early fractions was estimated using a modified Bradford microassay procedure (Bio-Rad) with bovine γ-globulin as the standard protein according to the manufacturer's directions. For the final glycerol gradient-purified fraction, the protein concentration was estimated by silver staining. All steps were carried out at 0–4 °C unless otherwise noted. A crude nuclear extract was fractionated by ammonium sulfate precipitation; the TIF-IB-containing fraction (0.5–1.82 m(NH4)2SO4) also contained the components needed for transcription of RNA polymerase III-transcribed genes (28Radebaugh C.A. Matthews J.L. Geiss G.K. Liu F. Wong J.-M. Bateman E. Camier S. Sentenac A. Paule M.R. Mol. Cell. Biol. 1994; 14: 597-605Crossref PubMed Scopus (43) Google Scholar, 38Lofquist A.K. Li H. Imboden M.A. Paule M.R. Nucleic Acids Res. 1993; 21: 3233-3238Crossref PubMed Scopus (23) Google Scholar), while RNA polymerase I was found in the 1.82–3.56m (NH4)2SO4 fraction (28Radebaugh C.A. Matthews J.L. Geiss G.K. Liu F. Wong J.-M. Bateman E. Camier S. Sentenac A. Paule M.R. Mol. Cell. Biol. 1994; 14: 597-605Crossref PubMed Scopus (43) Google Scholar). This fraction is called the “nuclear extract/pol III cut.” The TIF-IB-containing fraction from 225 g (wet weight) of A. castellanii cells containing 1700 mg of protein and 22.5 units of TIF-IB activity was dialyzed against 100 mm KCl in HEG20 (50 mm HEPES, pH 7.9, 0.2 mmEDTA, 20% glycerol, 1 mm dithiothreitol, 0.1 mm phenylmethanesulfonyl fluoride) to remove ammonium sulfate and convert the extract to 100 mm KCl. This was loaded onto a 50-ml DEAE-Sepharose fast flow column (2.5 × 10.2-cm) equilibrated to 100 mm KCl in HEG20 at a linear flow rate of 10 cm/h (0.5 column volume/h). This low flow rate is necessary to achieve optimal binding of TIF-IB to the exchange medium. The column was washed with 5 column volumes of HEG20 containing 100 mm KCl at a linear flow rate of 25 cm/h. The column was developed with a 5-column volume linear gradient from 100 to 500 mm KCl in HEG20 at 25 cm/h. Eighty fractions were collected and assayed for TIF-IB by a run-off transcription assay. TIF-IB reproducibly elutes with a peak at 280 mm KCl. RNA polymerase III is primarily in the unbound flow-through, but a small amount elutes ahead of and overlapping with the TIF-IB. The active fractions were pooled and diluted to 150 mm KCl in HEG20. The DEAE-Sepharose fast flow pool was loaded at a linear flow rate of 10 cm/h (2 column volumes/h) onto a 25-ml BioRex 70 column (2.5 × 5.1 cm) previously equilibrated with 150 mm KCl in HEG20. The column was washed with 5 column volumes of HEG20 containing 150 mm KCl at 15 cm/h. The column was developed with a linear gradient from 150 mm to 1000 mm KCl in HEG20 at a linear flow rate of 15 cm/h. Eighty fractions were collected and assayed as above. TIF-IB elutes with a peak at 330 mm KCl. The active fractions from the BioRex 70 column were pooled and dialyzed against H20EG10 (20 mm HEPES, pH 7.9, 0.2 mm EDTA, 10% glycerol, 1 mm dithiothreitol, 0.1 mm phenylmethanesulfonyl fluoride) containing 150 mm KCl. The pool was prepared for loading onto the DNA affinity column by adding MgCl2 to 10 mm, salmon sperm DNA to 0.09 mg/mg of protein, and sufficient 2-fold concentrated H20EG10 to maintain a constant buffer concentration. This final set of conditions was found to be optimal for binding to promoter DNA by EMSA. It is the same as the optimal set of conditions for transcription, except salmon sperm DNA was added to compete nonspecific DNA-binding proteins, and the KCl concentration was increased from 100 to 150 mm. The latter is allowable because of the point mutations, which specifically increase TIF-IB binding to the promoter (see “Preparation of Promoter-DNA-Sepharose 4B”). Five ml of promoter-DNA-Sepharose 4B was mixed with the prepared BioRex 70 pool and incubated with gentle agitation at room temperature (22 °C) for 1 h. The affinity medium was then loaded at 4 °C into a 1.5 × 2.8-cm column and the supernatant recirculated through the column at a linear flow rate of 5 cm/h until it had passed over the column three times. The column was washed with 5 column volumes of H20EG10containing 150 mm KCl, 10 mm MgCl2and 0.1% Nonidet P-40 (v/v) at 5 cm/h (1 column volume/h). The column was developed with a 5-column volume gradient from 150 to 1000 mm KCl in the same buffer at 5 cm/h. 40 fractions were collected and analyzed as above. TIF-IB eluted as a peak centered at 460 mm KCl. Promoter-DNA affinity-purified TIF-IB could be concentrated using a Microcon-10 microconcentrator according to the manufacturer's instructions (Amicon, Beverly, MA) or by diluting to 150 mm KCl with H20EG10, binding to a 1-ml (0.9 × 1.6 cm) BioRex-70 column and eluting with a step gradient of 600 mm KCl in H20EG10. 17.5–35% (v/v) glycerol gradients in 50 mm HEPES, pH 7.9, 0.2 mm EDTA, 1 mm dithiothreitol, 0.1 mm phenylmethanesulfonyl fluoride, 0.1% Nonidet P-40 (v/v), 100 mm KCl were prepared in 13 × 51-mm polyallomer centrifuge tubes. 200 μl of the concentrated promoter-DNA affinity column pool was layered on the top of each gradient and centrifuged for 15 h at 47,500 rpm in a Beckman SW50.1 rotor at 4 °C. The gradients were fractionated by pumping Fluorinert FC-40 (ISCO, Lincoln, NE) into the bottom of the tubes using an ISCO density gradient fractionator. 23 fractions were collected and assayed as above. The peak of TIF-IB is reproducibly in fractions 13 and 14, which is just above the position to which A. castellanii RNA polymerase I sediments in the same gradients (centered at fraction 15). The yield of TIF-IB is based upon the sum of the activities in the most active fractions. The amount of protein in these fractions is too low to estimate accurately using a modified Bradford assay. Instead, protein concentrations were estimated from silver staining and densitometric scanning. TIF-IB was purified from a nuclear extract using DEAE-fast flow, BioRex 70, and promoter-DNA affinity chromatography followed by rate zonal sedimentation in a glycerol gradient (Fig. 1 and Table I). Because there are inhibitors in the nuclear extract (28Radebaugh C.A. Matthews J.L. Geiss G.K. Liu F. Wong J.-M. Bateman E. Camier S. Sentenac A. Paule M.R. Mol. Cell. Biol. 1994; 14: 597-605Crossref PubMed Scopus (43) Google Scholar) leading to an apparent increase in activity after the first purification step, the DEAE-Sepharose fast flow fraction was assigned as 100% in Table I. The promoter-DNA affinity column alone purified TIF-IB 226-fold. The factor at this point is nearly homogeneous (Fig. 2). Even after the promoter-DNA affinity column step, the polypeptides that make up TIF-IB can be readily discerned (Fig. 2 B), along with a number of contaminating polypeptides that are present in variable amounts from preparation to preparation (cf. Fig. 2 B and Fig. 3 B, lane L). In the 7.5% gel shown in Fig. 2, the TBP is run off the bottom of the gel; however, the presence of TBP in the purified TIF-IB as well as in the promoter-DNA·TIF-IB complex has been demonstrated in previous studies (26Gong X. Radebaugh C.A. Geiss G.K. Simon M.N. Paule M.R. Mol. Cell. Biol. 1995; 15: 4956-4963Crossref PubMed Scopus (18) Google Scholar, 28Radebaugh C.A. Matthews J.L. Geiss G.K. Liu F. Wong J.-M. Bateman E. Camier S. Sentenac A. Paule M.R. Mol. Cell. Biol. 1994; 14: 597-605Crossref PubMed Scopus (43) Google Scholar). We have also found that multiple rounds of promoter-DNA affinity chromatography did not significantly improve the purity of TIF-IB. The glycerol gradient-purified TIF-IB had a specific activity of 211 units/mg of protein and was purified approximately 16,000-fold from the TIF-IB-containing nuclear extract, or 55,000-fold from the whole cells (39D'Alessio J.M. Spindler S.R. Paule M.R. J. Biol. Chem. 1979; 254: 4085-4091Abstract Full Text PDF PubMed Google Scholar).Table IPurification of TIF-IBFractionVolumeTotal protein1-aProtein concentrations were determined as described under “Experimental Procedures.”Total unitsSpecific activityPurificationYieldmlmgunits/mg-fold%Nuclear extract/pol III cut45170022.50.013DEAE-Sepharose Fast Flow6053044.00.0836.31001-bChosen as the starting point for yield calculation because of inhibitors in the nuclear extract.BioRex 70505638.70.6905388Promoter-DNA affinity7.50.2539.115912,00089Glycerol gradient0.080.1327.421116,000621-a Protein concentrations were determined as described under “Experimental Procedures.”1-b Chosen as the starting point for yield calculation because of inhibitors in the nuclear extract. Open table in a new tab Figure 2Elution profile of TIF-IB from the promoter-DNA affinity column. A, the specific run-off transcription product phosphor image and corresponding fraction number, load (L), and flow-through (FT) are indicated.B, a 7.5% silver-stained SDS-polyacrylamide gel of the fractions whose activities are shown in A. The relative molecular weights, in thousands, of the polypeptides identified to be components of TIF-IB are marked on the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Rate zonal sedimentation of A. castellanii TIF-IB in a glycerol gradient. A, the specific run-off transcription product phosphor image and corresponding fraction number. L, promoter-DNA affinity pool loaded onto the gradient. B, a 10% SDS-polyacrylamide gel of the fractions assayed in A, stained with Coomassie Blue. Molecular weights of the size marke" @default.
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