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• W2031496637 abstract "Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, τA and τB, using the baculovirus expression system. The B block-binding module, rτB, was reconstituted with rτ138, rτ91, and rτ60 subunits. rτ131, rτ95, and rτ55 formed also a stable complex, rτA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B″ fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system. Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, τA and τB, using the baculovirus expression system. The B block-binding module, rτB, was reconstituted with rτ138, rτ91, and rτ60 subunits. rτ131, rτ95, and rτ55 formed also a stable complex, rτA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B″ fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system. RNA polymerase III is responsible for the transcription of some 300 different genes in yeast, encoding mostly tRNAs (1Harismendy O. Gendrel C.G. Soularue P. Gidrol X. Sentenac A. Werner M. Lefebvre O. EMBO J. 2003; 22: 4738-4747Crossref PubMed Scopus (126) Google Scholar, 2Moqtaderi Z. Struhl K. Mol. Cell. Biol. 2004; 24: 4118-4127Crossref PubMed Scopus (128) Google Scholar, 3Roberts D.N. Stewart A.J. Huff J.T. Cairns B.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14695-14700Crossref PubMed Scopus (140) Google Scholar). Transcription by RNA polymerase III requires two general auxiliary factors, TFIIIC and TFIIIB, and a 5SRNA gene-specific factor, TFIIIA (reviewed in Ref. 4Geiduschek E.P. Kassavetis G.A. J. Mol. Biol. 2001; 310: 1-26Crossref PubMed Scopus (302) Google Scholar). The primary step in tRNA gene activation is the binding of TFIIIC to the intragenic promoter elements, the A and the B blocks. DNA-bound TFIIIC directs the assembly of TFIIIB, upstream of the transcription start site, and TFIIIB in turn recruits RNA polymerase III for multiple transcription cycles. Transcription of eukaryotic class III genes is a variation of this scheme that involves a cascade of protein-DNA and protein-protein interactions (4Geiduschek E.P. Kassavetis G.A. J. Mol. Biol. 2001; 310: 1-26Crossref PubMed Scopus (302) Google Scholar, 5Schramm L. Hernandez N. Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (438) Google Scholar, 6Chedin S. Ferri M.L. Peyroche G. Andrau J.C. Jourdain S. Lefebvre O. Werner M. Carles C. Sentenac A. Cold Spring Harb. Symp. Quant Biol. 1998; 63: 381-389Crossref PubMed Scopus (67) Google Scholar, 7Brown T.R. Scott P.H. Stein T. Winter A.G. White R.J. Gene Expr. 2000; 9: 15-28Crossref PubMed Scopus (33) Google Scholar). Yeast (Saccharomyces cerevisiae) TFIIIC is a multifunctional, multisubunit factor comprising six polypeptides organized in two large subassemblies, τA and τB. Identified by limited proteolysis and electron microscopy (8Marzouki N. Camier S. Ruet A. Moenne A. Sentenac A. Nature. 1986; 323: 176-178Crossref PubMed Scopus (49) Google Scholar, 9Schultz P. Marzouki N. Marck C. Ruet A. Oudet P. Sentenac A. EMBO J. 1989; 8: 3815-3824Crossref PubMed Scopus (49) Google Scholar), τB binds tightly to the B block that is located at a variable distance from the start site. Biochemical and genetic evidences indicated that τB likely comprises three subunits, τ138, τ 91, and τ 60 (10Gabrielsen O.S. Marzouki N. Ruet A. Sentenac A. Fromageot P. J. Biol. Chem. 1989; 264: 7505-7511Abstract Full Text PDF PubMed Google Scholar, 11Arrebola R. Manaud N. Rozenfeld S. Marsolier M.C. Lefebvre O. Carles C. Thuriaux P. Conesa C. Sentenac A. Mol. Cell. Biol. 1998; 18: 1-9Crossref PubMed Google Scholar, 12Deprez E. Arrebola R. Conesa C. Sentenac A. Mol. Cell. Biol. 1999; 19: 8042-8051Crossref PubMed Scopus (31) Google Scholar, 13Lefebvre O. Ruth J. Sentenac A. J. Biol. Chem. 1994; 269: 23374-23381Abstract Full Text PDF PubMed Google Scholar). Although τ138 and τ91 cooperate in B block binding (11Arrebola R. Manaud N. Rozenfeld S. Marsolier M.C. Lefebvre O. Carles C. Thuriaux P. Conesa C. Sentenac A. Mol. Cell. Biol. 1998; 18: 1-9Crossref PubMed Google Scholar), τ 60 appears to link τA and τB domains and, quite unexpectedly, to participate in TBP recruitment (12Deprez E. Arrebola R. Conesa C. Sentenac A. Mol. Cell. Biol. 1999; 19: 8042-8051Crossref PubMed Scopus (31) Google Scholar, 14Mylona A. Acker J. Fernandez-Tornero C. Sentenac A. Muller C.W. Protein Expr. Purif. 2005; 45: 255-261Crossref PubMed Scopus (2) Google Scholar). The τA domain, visualized by electron microscopy, probably comprises τ 95 and τ 55, which are thought to participate in A block binding (15Braun B.R. Bartholomew B. Kassavetis G.A. Geiduschek E.P. J. Mol. Biol. 1992; 228: 1063-1077Crossref PubMed Scopus (72) Google Scholar, 16Bartholomew B. Kassavetis G.A. Braun B.R. Geiduschek E.P. EMBO J. 1990; 9: 2197-2205Crossref PubMed Scopus (136) Google Scholar), and τ131, which is mostly responsible for TFIIIB assembly (17Khoo B. Brophy B. Jackson S.P. Genes Dev. 1994; 8: 2879-2890Crossref PubMed Scopus (108) Google Scholar, 18Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). τ131 is the only subunit of TFIIIC extending upstream of the start site (19Bartholomew B. Kassavetis G.A. Geiduschek E.P. Mol. Cell. Biol. 1991; 11: 5181-5189Crossref PubMed Scopus (125) Google Scholar). All six genes of yeast TFIIIC have been cloned and found to be essential for yeast cell viability, as was each of the 17 subunits of RNA polymerase III and the three components of TFIIIB (6Chedin S. Ferri M.L. Peyroche G. Andrau J.C. Jourdain S. Lefebvre O. Werner M. Carles C. Sentenac A. Cold Spring Harb. Symp. Quant Biol. 1998; 63: 381-389Crossref PubMed Scopus (67) Google Scholar). TFIIIB is a multiprotein transcription factor comprising three polypeptides that do not form a stable complex when not bound to DNA (20Kassavetis G.A. Bartholomew B. Blanco J.A. Johnson T.E. Geiduschek E.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7308-7312Crossref PubMed Scopus (102) Google Scholar, 21Huet J. Conesa C. Manaud N. Chaussivert N. Sentenac A. Nucleic Acids Res. 1994; 22: 3433-3439Crossref PubMed Scopus (30) Google Scholar). It can be chromatographically separated into two subfractions, B′ containing the TATA-binding factor TBP and Brf1 and B″ containing Bdp1. TFIIIC-dependent TFIIIB assembly onto TATA-less genes involves a stepwise series of interactions and conformational changes starting with the recruitment of Brf1 by τ131, the entry of TBP mediated by Brf1 and probably τ60, followed by the binding of Bdp1, directed by τ131, that stabilizes and locks the TFIIIB-DNA complex (12Deprez E. Arrebola R. Conesa C. Sentenac A. Mol. Cell. Biol. 1999; 19: 8042-8051Crossref PubMed Scopus (31) Google Scholar, 20Kassavetis G.A. Bartholomew B. Blanco J.A. Johnson T.E. Geiduschek E.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7308-7312Crossref PubMed Scopus (102) Google Scholar, 22Kassavetis G.A. Joazeiro C.A. Pisano M. Geiduschek E.P. Colbert T. Hahn S. Blanco J.A. Cell. 1992; 71: 1055-1064Abstract Full Text PDF PubMed Scopus (182) Google Scholar). TFIIIB is able to assemble autonomously in vitro, via the interaction of TBP with the strong TATA box of the SNR6 gene (23Moenne A. Camier S. Anderson G. Margottin F. Beggs J. Sentenac A. EMBO J. 1990; 9: 271-277Crossref PubMed Scopus (67) Google Scholar, 24Whitehall S.K. Kassavetis G.A. Geiduschek E.P. Genes Dev. 1995; 9: 2974-2985Crossref PubMed Scopus (59) Google Scholar), but TFIIIC is required in vivo to transcribe the few TATA-containing class III genes (1Harismendy O. Gendrel C.G. Soularue P. Gidrol X. Sentenac A. Werner M. Lefebvre O. EMBO J. 2003; 22: 4738-4747Crossref PubMed Scopus (126) Google Scholar, 25Brow D.A. Guthrie C. Genes Dev. 1990; 4: 1345-1356Crossref PubMed Scopus (108) Google Scholar, 26Eschenlauer J.B. Kaiser M.W. Gerlach V.L. Brow D.A. Mol. Cell. Biol. 1993; 13: 3015-3026Crossref PubMed Scopus (79) Google Scholar). This paradox was resolved by the observation that TFIIIC relieves chromatin repression in vitro (27Burnol A.F. Margottin F. Huet J. Almouzni G. Prioleau M.N. Mechali M. Sentenac A. Nature. 1993; 362: 475-477Crossref PubMed Scopus (87) Google Scholar, 28Marsolier M.C. Chaussivert N. Lefebvre O. Conesa C. Werner M. Sentenac A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11938-11942Crossref PubMed Scopus (19) Google Scholar), and in vivo (28Marsolier M.C. Chaussivert N. Lefebvre O. Conesa C. Werner M. Sentenac A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11938-11942Crossref PubMed Scopus (19) Google Scholar). However, yeast TFIIIC did not show any detectable histone acetyltransferase activity (6Chedin S. Ferri M.L. Peyroche G. Andrau J.C. Jourdain S. Lefebvre O. Werner M. Carles C. Sentenac A. Cold Spring Harb. Symp. Quant Biol. 1998; 63: 381-389Crossref PubMed Scopus (67) Google Scholar), at variance with purified human TFIIIC (29Hsieh Y.J. Kundu T.K. Wang Z. Kovelman R. Roeder R.G. Mol. Cell. Biol. 1999; 19: 7697-7704Crossref PubMed Scopus (88) Google Scholar, 30Kundu T.K. Wang Z. Roeder R.G. Mol. Cell. Biol. 1999; 19: 1605-1615Crossref PubMed Scopus (107) Google Scholar).The basal transcription system described above directs accurate initiation and termination of transcription in vitro on a variety of class III genes (31Ruth J. Conesa C. Dieci G. Lefebvre O. Dusterhoft A. Ottonello S. Sentenac A. EMBO J. 1996; 15: 1941-1949Crossref PubMed Scopus (77) Google Scholar, 32Kassavetis G.A. Nguyen S.T. Kobayashi R. Kumar A. Geiduschek E.P. Pisano M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9786-9790Crossref PubMed Scopus (91) Google Scholar). It is still possible, however, that additional components may be needed to reach the high transcription rates observed in vivo. Indeed, efficient transcription of the SNR6 gene, which has a degenerate A block and a distant B block located downstream of the termination signal, was shown to require the Nhp6 proteins in vivo and in vitro (33Kruppa M. Moir R.D. Kolodrubetz D. Willis I.M. Mol. Cell. 2001; 7: 309-318Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 34Lopez S. Livingstone-Zatchej M. Jourdain S. Thoma F. Sentenac A. Marsolier M.C. Mol. Cell. Biol. 2001; 21: 3096-3104Crossref PubMed Scopus (44) Google Scholar). There was also the intriguing observation that recombinant Bdp1 directed accurate transcription of the SUP4 tRNA gene at a low level and needed to be supplemented with TFIIIE to fully restore the transcription level obtained with purified B″ fraction (31Ruth J. Conesa C. Dieci G. Lefebvre O. Dusterhoft A. Ottonello S. Sentenac A. EMBO J. 1996; 15: 1941-1949Crossref PubMed Scopus (77) Google Scholar). TFIIIE factor activity has not yet been characterized, and its mode of action is still unclear (35Dieci G. Duimio L. Coda-Zabetta F. Sprague K.U. Ottonello S. J. Biol. Chem. 1993; 268: 11199-11207Abstract Full Text PDF PubMed Google Scholar).Ultimately, future advances in the definition and analyses of the RNA polymerase III transcription system will require its reconstitution with recombinant proteins. As a step toward this goal, we describe here the reconstitution of functional TFIIIC by expression of its subunits in insect cells. The τA and the τB domains of the factor could be produced independently and analyzed. The transcription system reconstituted with recombinant TFIIIC, recombinant TFIIIB, and highly purified RNA polymerase III directed a level of specific transcription similar to the one obtained with affinity-purified endogenous TFIIIC.MATERIALS AND METHODSProduction and Expression of Recombinant Baculoviruses—The open reading frame of FLAG-τ138, HA-τ 95, τ 91, τ 60, τ 55, or Brf1–His were inserted in PVL1392 vector (Pharmingen) and then recombined with baculovirus DNA (Bacvector 3000 DNA; Novagen) in Spodoptera frugiperda (SF9 cells). The recombinant viruses were plaque-purified, and viral stocks were prepared by three-step growth amplifications. The open reading frames of τ138, His-τ131, and τ 60-His were subcloned in a pFastbac1 vector (Invitrogen). The resulting plasmids were used for bacmid production according to the manufacturer's protocol (Invitrogen). High Five cells (typically 2 × 107 cells) were infected with one baculovirus or co-infected with combinations of recombinant baculoviruses (from two to six) as indicated. Multiplicities of infection were adjusted so as to balance the amount of recombinant proteins simultaneously expressed from each virus. The cells were collected 72 h postinfection. The protein extracts were prepared as described (36Dumay-Odelot H. Acker J. Arrebola R. Sentenac A. Marck C. Mol. Cell. Biol. 2002; 22: 298-308Crossref PubMed Scopus (21) Google Scholar).Purification of the Recombinant Proteins—Preparation of rτA. GST-τ131 4The abbreviations used are: GST, glutathione S-transferase; HA, hemagglutinin; AS, ammonium sulfate; Pol, polymerase; PAP, potato acid phosphatase; Pipes, 1,4-piperazinediethanesulfonic acid; TBP, TATA-binding protein. (36Dumay-Odelot H. Acker J. Arrebola R. Sentenac A. Marck C. Mol. Cell. Biol. 2002; 22: 298-308Crossref PubMed Scopus (21) Google Scholar), HA-τ95 and τ55 were co-expressed in High Five cells. The three polypeptides were co-purified successively by anti-HA-tag affinity column (Sigma) followed by GSH affinity column (Amersham Biosciences) chromatography to yield the rτA subcomplex (according to the manufacturer's instructions).Preparation of rτB—High Five cell extract co-expressing FLAG-τ138, τ91, and τ60-His was subjected to chromatography on heparin Hyper D (Biosepra) equilibrated in 50 mm Tris, pH 7.5, 100 mm NaCl, 20% glycerol, 5 mm β-mercaptoethanol, and protease inhibitor mixture (Complete™; Roche Applied Science). The proteins were eluted with a 30-column volume linear gradient of NaCl from 0.1 to 1 m. The fractions were then tested in gel shift assay using tDNA3Leu gene as a probe (see below). rTFIIICa and rTFIIICb were purified from High Five cells (2 × 109 cells) co-expressing τ138, His-τ131, HA-τ95, τ91, τ60, and τ55, using the Sprint Biocad system (Applied Biosystem) at 10 ml/min. The extracts, prepared in buffer B0 (50 mm Tris, pH 8, 20% glycerol, 5 mm β-mercaptoethanol, and protease inhibitor mixture) containing 40 mm ammonium sulfate (AS), were first adjusted to 250 mm AS and then loaded onto a 35-ml heparin HyperD column previously equilibrated with the same buffer. The resin was washed with 10 column volumes of buffer B0 containing 360 mm AS. A 15-column volume linear gradient of AS from 360 to 750 mm was then applied. Fractions were collected and assayed for TFIIIC-DNA binding activity (see below). rTFIIICa (fractions 30–40) or rTFIIICb (fractions 60–80) were pooled separately, adjusted to 550 mm AS, and subjected to fast liquid chromatography in a 0.8-ml Poros MC 20 (Applied Biosystem) column charged with Cobalt. rTFIIICa or rTFIIICb were eluted with buffer B0 containing 40 mm AS and 300 mm imidazole (pH adjusted to 8).His-TBP (36Dumay-Odelot H. Acker J. Arrebola R. Sentenac A. Marck C. Mol. Cell. Biol. 2002; 22: 298-308Crossref PubMed Scopus (21) Google Scholar), and His-Brf1 were expressed in High Five cells and purified successively by metal chelate chromatography on Poros MC 20 loaded with nickel and heparin chromatography (Poros 20 HE; Applied Biosystem). rBdp1 was prepared as described (37Ferrari R. Rivetti C. Acker J. Dieci G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13442-13447Crossref PubMed Scopus (47) Google Scholar).DNA Binding and in Vitro Transcription Assays—TFIIIC-DNA interactions were monitored by gel shift assays as described previously (38Jourdain S. Acker J. Ducrot C. Sentenac A. Lefebvre O. J. Biol. Chem. 2003; 278: 10450-10457Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) using a 32P-labeled DNA fragment carrying the tRNA3Leu or the SUP4 tRNATyr gene as a probe. The amounts of proteins used in the various gel shift assays were as follows: affinity-purified rTFIIIC (10 ng), heparin-purified rTFIIIC (50 ng), heparin-purified rτB (30 ng), and Mono Q endogenous TFIIIC (100 ng). The final KCl concentration was adjusted to 180 mm instead of 120 mm (used with TFIIIC) when rτB was assayed. The limited proteolysis assays were performed as described (12Deprez E. Arrebola R. Conesa C. Sentenac A. Mol. Cell. Biol. 1999; 19: 8042-8051Crossref PubMed Scopus (31) Google Scholar), using 50 ng of heparin-purified rτB or 150 ng of Mono Q-purified endogenous TFIIIC (38Jourdain S. Acker J. Ducrot C. Sentenac A. Lefebvre O. J. Biol. Chem. 2003; 278: 10450-10457Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The apparent dissociation constant (Kapp) of rTFIIICa-, rTFIIICb-, or yTFIIIC-tDNA3Leu complexes was determined as described previously (38Jourdain S. Acker J. Ducrot C. Sentenac A. Lefebvre O. J. Biol. Chem. 2003; 278: 10450-10457Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar).Standard in vitro transcriptions were performed as previously described (12Deprez E. Arrebola R. Conesa C. Sentenac A. Mol. Cell. Biol. 1999; 19: 8042-8051Crossref PubMed Scopus (31) Google Scholar, 39Huet J. Conesa C. Carles C. Sentenac A. J. Biol. Chem. 1997; 272: 18341-18349Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), using the following amounts of proteins: 20 ng of affinity-purified rTFIIIC (or 100 ng of Mono Q-purified endogenous yTFIIIC), 0.5 μg of partially purified B″ fraction (22Kassavetis G.A. Joazeiro C.A. Pisano M. Geiduschek E.P. Colbert T. Hahn S. Blanco J.A. Cell. 1992; 71: 1055-1064Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 37Ferrari R. Rivetti C. Acker J. Dieci G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13442-13447Crossref PubMed Scopus (47) Google Scholar), or 10 ng of pure rBdp1 when indicated, 20 ng of rTBP, 10 ng of rBrf1, 100 ng of highly purified RNA pol III and 100 ng of the indicated DNA plasmid templates. As estimated by SDS-PAGE analysis followed by Coomassie Blue staining, 0.5 μg of the crude B″ fraction used provide no more than 20 ng of Bdp1 polypeptide. When 5 S RNA gene was transcribed, 40 ng of purified rTFIIIA (40Ottonello S. Ballabeni A. Soncini C. Dieci G. Biochem. Biophys. Res. Commun. 1994; 203: 1217-1223Crossref PubMed Scopus (10) Google Scholar) was added to the transcription mixture. The transcriptions reactions were allowed to proceed for 45 min at 25 °C, and the transcripts were analyzed by electrophoresis on 6% polyacrylamide, 8 m urea gel.To analyze the initiation of transcription, a 17-mer assay was performed as described previously (41Chedin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (151) Google Scholar). Stable ternary complexes were formed by incubating the transcriptions proteins (same amount as for the standard in vitro transcription presented in this study) for 20 min at 25 °C. Purified RNA Pol III, ATP, CTP, and α-32P-labeled UTP were then added, and the transcription was allowed to proceed for 20 min at 25 °C. The reaction products were separated by electrophoresis on 15% polyacrylamide, 8 m urea gel.Footprint—Binding reactions were calibrated using heparin-purified rτB or Mono Q-purified endogenous TFIIIC (38Jourdain S. Acker J. Ducrot C. Sentenac A. Lefebvre O. J. Biol. Chem. 2003; 278: 10450-10457Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) to obtain a complete retardation of the probe. The TFIIIC-DNA complexes obtained were then subjected to DNase protection as described (42Camier S. Gabrielsen O. Baker R. Sentenac A. EMBO J. 1985; 4: 491-500Crossref PubMed Scopus (49) Google Scholar), and the DNA fragments were resolved on an 8% polyacrylamide sequencing gel.Phosphatase Treatments—Potato acid phosphatase (PAP; Fluka) in ammonium sulfate suspension was centrifuged for 30 min at 15,000 rpm at 4 °C and dissolved in PPA buffer (10 mm Pipes, pH 6, 100 mm NaCl, and 3mm MgCl2) at a final concentration of 0.5 unit/μl. 50 ng of heparin-purified rTFIIIC, 15 ng of affinity-purified rTFIIIC, or 150 ng of Mono Q purified endogenous yTFIIIC were incubated with increasing amounts of PAP (from 0.025 to 0.75 unit) as indicated, or bovine serum albumin in PPA buffer for 30 min at 30 °C. Phosphatase inhibitor mixture II (Sigma) was then added at a 1:20 dilution to the reaction mixtures before DNA binding or in vitro transcription assays.Southwestern Blot—The Southwestern blot was performed as previously described (39Huet J. Conesa C. Carles C. Sentenac A. J. Biol. Chem. 1997; 272: 18341-18349Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Briefly 5–10 μg of each purified protein was subjected to 8% SDS-PAGE and blotted to nitrocellulose. The filters were first washed with the a buffer containing 20 mm Hepes, pH 7.5, 0.1 mm EDTA, 5 mm MgCl2, 100 mm KCl and then incubated for 30 min with 20 mm phosphate-buffered saline, pH 7.2, containing 2.5% (v/v) Nonidet P-40, 1% (w/v) gelatin, 40 mm NaCl, 0.5 mm EDTA, and 10% (v/v) glycerol). After a 30-min prehybridization period with 20 mm phosphate-buffered saline, pH 7.2, containing 40 mm NaCl, 0.05% (w/v) gelatin, the filters were then incubated for 1 h at 4°C with the same buffer C in the presence of a 32P-labeled (106 cpm/ml) alternating copolymer poly(dA-dT) from Sigma. Finally, the membrane was washed three times for 5 min with binding buffer, and the labeled polypeptides were revealed by autoradiography.RESULTSCharacterization of the Two Structural Domains of TFIIIC—To attempt the in vivo assembly of yeast TFIIIC, we constructed various recombinant baculoviruses for directing the production of each individual TFIIIC subunit in insect cells. The six subunits were overexpressed quite efficiently, without much noticeable proteolysis (data not shown). Some subunits were epitope-tagged to allow affinity purification of protein complexes. In co-expression experiments to produce partial or complete TFIIIC, insect cells were co-infected with appropriate amounts of viruses so as to co-express a similar level of each subunit. Next, we looked for protein assemblies that could form stable complexes with tDNA. High Five cells were co-infected with various combinations of recombinant baculoviruses to produce the six polypeptides (τ138, GST-τ131, HA-τ95, τ91, τ60, and τ55) or all possible combinations of five subunits. All of the polypeptides were present in similar amounts in the different extracts, as shown by immunoblotting (Fig. 1).In gel retardation assays using crude cell extracts, two complexes of different migration rates were detected (data not shown). The larger one was only present when all six subunits were co-expressed, whereas the quickly migrating one was detected when τ138, τ91, and τ60 were coexpressed (data not shown). This complex was likely related to the τB protease-resistant complex previously characterized with TFIIIC preparations subjected to limited proteolysis (8Marzouki N. Camier S. Ruet A. Moenne A. Sentenac A. Nature. 1986; 323: 176-178Crossref PubMed Scopus (49) Google Scholar). Indeed, τ138 was shown to be part of the protease-resistant τB domain (10Gabrielsen O.S. Marzouki N. Ruet A. Sentenac A. Fromageot P. J. Biol. Chem. 1989; 264: 7505-7511Abstract Full Text PDF PubMed Google Scholar), and biochemical studies have suggested that τ91 and τ60 also belonged to this DNA-binding subcomplex (11Arrebola R. Manaud N. Rozenfeld S. Marsolier M.C. Lefebvre O. Carles C. Thuriaux P. Conesa C. Sentenac A. Mol. Cell. Biol. 1998; 18: 1-9Crossref PubMed Google Scholar, 12Deprez E. Arrebola R. Conesa C. Sentenac A. Mol. Cell. Biol. 1999; 19: 8042-8051Crossref PubMed Scopus (31) Google Scholar, 16Bartholomew B. Kassavetis G.A. Braun B.R. Geiduschek E.P. EMBO J. 1990; 9: 2197-2205Crossref PubMed Scopus (136) Google Scholar). We therefore attempted to reconstitute the minimal rτB module using full-length polypeptides. Insect protein extracts containing recombinant FLAG-τ138, τ91, or τ60-His expressed either alone or in combination were partially purified by chromatography on a heparin column, and the fractions, eluted by a salt gradient, were analyzed by gel shift assays (Fig. 2A and data not shown). No protein-DNA complex was formed using the fractions from a control cell extract, indicating that no insect proteins were able to form a stable complex with the yeast tRNA probe under the rather stringent binding conditions used (125 mm KCl and 300 ng of competitor DNA; data not shown). Using extracts expressing one subunit, we only detected DNA binding activity with τ91. The nonspecific DNA binding activity of τ91 was previously reported (11Arrebola R. Manaud N. Rozenfeld S. Marsolier M.C. Lefebvre O. Carles C. Thuriaux P. Conesa C. Sentenac A. Mol. Cell. Biol. 1998; 18: 1-9Crossref PubMed Google Scholar). Although τ91 was found to cooperate with τ138 for DNA binding (11Arrebola R. Manaud N. Rozenfeld S. Marsolier M.C. Lefebvre O. Carles C. Thuriaux P. Conesa C. Sentenac A. Mol. Cell. Biol. 1998; 18: 1-9Crossref PubMed Google Scholar), these subunits did not appear to assemble strongly (data not shown). On the contrary, τ91 and τ60 can form a stable complex that could represent the scaffold of the τB subcomplex (14Mylona A. Acker J. Fernandez-Tornero C. Sentenac A. Muller C.W. Protein Expr. Purif. 2005; 45: 255-261Crossref PubMed Scopus (2) Google Scholar). However, no specific DNA binding activity could be detected with this pair of subunits. In fact, the three subunits, τ138, τ91, and τ60, were necessary to reconstitute a strongly DNA-binding complex (Fig. 2A). In the absence of any of these three subunits, no other DNA-protein complex similar in size could be detected (data not shown). To demonstrate the presence of these three polypeptides in the B block-binding complex, rτB-tDNA3Leu complexes were incubated for 1 h at 25°C with increasing amounts of subunit-specific antibodies and analyzed by electrophoresis on a 5% polyacrylamide gel. As shown in Fig. 2B, anti-FLAG and anti-histidine monoclonal antibodies altered the migration of the rτB-tDNA complex (Fig. 2B, compare lane 1 with lanes 3, 4, 7, and 8). Anti-τ91 polyclonal antibodies also interfered with complex formation (Fig. 2B, lanes 5 and 6), as observed when endogenous TFIIIC (yTFIIIC) is used in gel shift assays (11Arrebola R. Manaud N. Rozenfeld S. Marsolier M.C. Lefebvre O. Carles C. Thuriaux P. Conesa C. Sentenac A. Mol. Cell. Biol. 1998; 18: 1-9Crossref PubMed Google Scholar). On the other hand, rτB-tDNA complex was not affected by control anti-T7 antibodies (Fig. 2B, compare lanes 1 and 2). These results indicated that τ138, τ91, and τ60 reconstituted rτB. rτB-DNA interaction was then analyzed by DNA footprinting and compared with the characteristic footprint observed with yTFIIIC over the tRNA3Leu gene (43Kassavetis G.A. Riggs D.L. Negri R. Nguyen L.H. Geiduschek E.P. Mol. Cell. Biol. 1989; 9: 2551-2566Crossref PubMed Scopus (185) Google Scholar) (Fig. 2C, compare lanes 3 and 1). As expected, rτB gave a partial footprint, spanning only the 3′ half of the gene, over the B block. The protection of the B block region was similar with rτB and yTFIIIC (Fig. 2C, compare lanes 2 and 3), which confirmed the binding specificity of the rτB complex. Limited α-chymotrypsin proteolysis of rτB generated a stable protein-tDNA3Leu complex of the same increased electrophoretic mobility as the protease-resistant τB complex (8Marzouki N. Camier S. Ruet A. Moenne A. Sentenac A. Nature. 1986; 323: 176-178Crossref PubMed Scopus (49) Google Scholar) generated from the endogenous yeast TFIIIC (Fig. 2D, compare lanes 3 and 5). Therefore, when bound to tDNA, the reconstituted rτB and the τB domain of TFIIIC appeared to have the same accessibility to the protease, supporting the model of a transcription factor made of two structural modules. Together, these data demonstrate that, using full-length polypeptides, the minimum specific B block-binding domain is composed of τ138, τ91, and τ60.FIGURE 2Reconstitution of rτB. High Five cells were infected with recombinant baculoviruses encoding FLAG-τ138, τ91, and His-τ60. Protein extracts were chromatographed on a heparin column, and bound proteins were eluted using an NaCl gradient as described under “Materials and Methods.” Protein-DNA complexes were analyzed by electrophoresis and autoradiography. A, gel shift assay. Heparin-purified fractions were incubated with a labeled DNA fragment harboring the tRNA3Leu gene. The position of rτB-DNA complexes is indicated on the left. B, polypeptide composition of rτB. Preformed rτB-tDNA3Leu complexes (" @default.
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• W2031496637 title "Reconstitution of the Yeast RNA Polymerase III Transcription System with All Recombinant Factors" @default.
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