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Originally published In Press as doi:10.1074/jbc.M600101200 on March 3, 2006

J. Biol. Chem., Vol. 281, Issue 17, 11685-11692, April 28, 2006
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Reconstitution of the Yeast RNA Polymerase III Transcription System with All Recombinant Factors*

Cécile Ducrot, Olivier Lefebvre, Emilie Landrieux1, Josée Guirouilh-Barbat2, André Sentenac, and Joel Acker3

From the Service de Biochimie et de Génétique Moléculaire, Bâtiment 144, CEA/Saclay, F-91191 Gif-sur-Yvette Cedex, France

Received for publication, January 5, 2006 , and in revised form, February 23, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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, {tau}A and {tau}B, using the baculovirus expression system. The B block-binding module, r{tau}B, was reconstituted with r{tau}138, r{tau}91, and r{tau}60 subunits. r{tau}131, r{tau}95, and r{tau}55 formed also a stable complex, r{tau}A, 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RNA polymerase III is responsible for the transcription of some 300 different genes in yeast, encoding mostly tRNAs (1–3). Transcription by RNA polymerase III requires two general auxiliary factors, TFIIIC and TFIIIB, and a 5SRNA gene-specific factor, TFIIIA (reviewed in Ref. 4). 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 (4–7). Yeast (Saccharomyces cerevisiae) TFIIIC is a multifunctional, multisubunit factor comprising six polypeptides organized in two large subassemblies, {tau}A and {tau}B. Identified by limited proteolysis and electron microscopy (8, 9), {tau}B binds tightly to the B block that is located at a variable distance from the start site. Biochemical and genetic evidences indicated that {tau}B likely comprises three subunits, {tau}138, {tau} 91, and {tau} 60 (10–13). Although {tau}138 and {tau}91 cooperate in B block binding (11), {tau} 60 appears to link {tau}A and {tau}B domains and, quite unexpectedly, to participate in TBP recruitment (12, 14). The {tau}A domain, visualized by electron microscopy, probably comprises {tau} 95 and {tau} 55, which are thought to participate in A block binding (15, 16), and {tau}131, which is mostly responsible for TFIIIB assembly (17, 18). {tau}131 is the only subunit of TFIIIC extending upstream of the start site (19). 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 (6). TFIIIB is a multiprotein transcription factor comprising three polypeptides that do not form a stable complex when not bound to DNA (20, 21). 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 {tau}131, the entry of TBP mediated by Brf1 and probably {tau}60, followed by the binding of Bdp1, directed by {tau}131, that stabilizes and locks the TFIIIB-DNA complex (12, 20, 22). TFIIIB is able to assemble autonomously in vitro, via the interaction of TBP with the strong TATA box of the SNR6 gene (23, 24), but TFIIIC is required in vivo to transcribe the few TATA-containing class III genes (1, 25, 26). This paradox was resolved by the observation that TFIIIC relieves chromatin repression in vitro (27, 28), and in vivo (28). However, yeast TFIIIC did not show any detectable histone acetyltransferase activity (6), at variance with purified human TFIIIC (29, 30).

The basal transcription system described above directs accurate initiation and termination of transcription in vitro on a variety of class III genes (31, 32). 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 (33, 34). 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 (31). TFIIIE factor activity has not yet been characterized, and its mode of action is still unclear (35).

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 {tau}A and the {tau}B 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 METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Production and Expression of Recombinant Baculoviruses—The open reading frame of FLAG-{tau}138, HA-{tau} 95, {tau} 91, {tau} 60, {tau} 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 {tau}138, His-{tau}131, and {tau} 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 x 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 (36).

Purification of the Recombinant Proteins—Preparation of r{tau}A. GST-{tau}1314 (36), HA-{tau}95 and {tau}55 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 r{tau}A subcomplex (according to the manufacturer's instructions).

Preparation of r{tau}B—High Five cell extract co-expressing FLAG-{tau}138, {tau}91, and {tau}60-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 beta-mercaptoethanol, and protease inhibitor mixture (CompleteTM; 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 Formula gene as a probe (see below). rTFIIICa and rTFIIICb were purified from High Five cells (2 x 109 cells) co-expressing {tau}138, His-{tau}131, HA-{tau}95, {tau}91, {tau}60, and {tau}55, 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 beta-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 (36), 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 (37).

DNA Binding and in Vitro Transcription Assays—TFIIIC-DNA interactions were monitored by gel shift assays as described previously (38) using a 32P-labeled DNA fragment carrying the Formula 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 r{tau}B (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 r{tau}B was assayed. The limited proteolysis assays were performed as described (12), using 50 ng of heparin-purified r{tau}B or 150 ng of Mono Q-purified endogenous TFIIIC (38). The apparent dissociation constant (Kapp) of rTFIIICa-, rTFIIICb-, or yTFIIIC-tDNA3Leu complexes was determined as described previously (38).

Standard in vitro transcriptions were performed as previously described (12, 39), 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 (22, 37), 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 (40) 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.


Figure 1
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  		FIGURE 1.
Co-expression of TFIIIC subunits. High Five cells were co-infected with recombinant baculoviruses expressing the six subunits of TFIIIC (lane 7) or only five of them (lanes 1–6, the missing polypeptide is indicated at the top). Total protein extracts (15 µg), were analyzed by Western blotting using antibodies specific to each TFIIIC subunit as indicated on the right.

 
To analyze the initiation of transcription, a 17-mer assay was performed as described previously (41). 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 {alpha}-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 r{tau}B or Mono Q-purified endogenous TFIIIC (38) to obtain a complete retardation of the probe. The TFIIIC-DNA complexes obtained were then subjected to DNase protection as described (42), 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 (39). 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.


Figure 2
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  		FIGURE 2.
Reconstitution of r{tau}B. High Five cells were infected with recombinant baculoviruses encoding FLAG-{tau}138, {tau}91, and His-{tau}60. 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 Formula gene. The position of r{tau}B-DNA complexes is indicated on the left. B, polypeptide composition of r{tau}B. Preformed r{tau}B-Formula complexes (lane 1) were incubated with 1µg of control anti-T7 (lane 2), 0.3 or 1 µg of anti-Histidine (lanes 3 and 4, respectively), anti-{tau}91 (lanes 5 and 6), or anti-FLAG (lanes 7 and 8) antibodies. C,r{tau}B interacts with the B block of tDNA. r{tau}B (lane 2) and endogenous yTFIIIC (lane 3) were examined for their footprint on a DNA fragment encompassing the Formula gene (schematized on the left, with the location of the A and B blocks and of the initiation site) as described under "Materials and Methods." Lane 1, control without protein. D, limited proteolysis of{tau}B. Heparin-purified r{tau}B(lanes 2 and 3) or endogenous Mono Q-purified yTFIIIC (lanes 4 and 5) were complexed with the Formula gene and then subjected to limited proteolysis (lanes 3 and 5) or not (lanes 2 and 4) by incubation with 10 ng of{alpha}-chymotrypsin for 10 min at 25 °C. Lane 1, DNA probe alone.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization 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 ({tau}138, GST-{tau}131, HA-{tau}95, {tau}91, {tau}60, and {tau}55) 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 {tau}138, {tau}91, and {tau}60 were coexpressed (data not shown). This complex was likely related to the {tau}B protease-resistant complex previously characterized with TFIIIC preparations subjected to limited proteolysis (8). Indeed, {tau}138 was shown to be part of the protease-resistant {tau}B domain (10), and biochemical studies have suggested that {tau}91 and {tau}60 also belonged to this DNA-binding subcomplex (11, 12, 16). We therefore attempted to reconstitute the minimal r{tau}B module using full-length polypeptides. Insect protein extracts containing recombinant FLAG-{tau}138, {tau}91, or {tau}60-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 {tau}91. The nonspecific DNA binding activity of {tau}91 was previously reported (11). Although {tau}91 was found to cooperate with {tau}138 for DNA binding (11), these subunits did not appear to assemble strongly (data not shown). On the contrary, {tau}91 and {tau}60 can form a stable complex that could represent the scaffold of the {tau}B subcomplex (14). However, no specific DNA binding activity could be detected with this pair of subunits. In fact, the three subunits, {tau}138, {tau}91, and {tau}60, 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, r{tau}B-Formula 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 r{tau}B-tDNA complex (Fig. 2B, compare lane 1 with lanes 3, 4, 7, and 8). Anti-{tau}91 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 (11). On the other hand, r{tau}B-tDNA complex was not affected by control anti-T7 antibodies (Fig. 2B, compare lanes 1 and 2). These results indicated that {tau}138, {tau}91, and {tau}60 reconstituted r{tau}B. r{tau}B-DNA interaction was then analyzed by DNA footprinting and compared with the characteristic footprint observed with yTFIIIC over the Formula gene (43) (Fig. 2C, compare lanes 3 and 1). As expected, r{tau}B 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 r{tau}B and yTFIIIC (Fig. 2C, compare lanes 2 and 3), which confirmed the binding specificity of the r{tau}B complex. Limited {alpha}-chymotrypsin proteolysis of r{tau}B generated a stable protein-tDNA3Leu complex of the same increased electrophoretic mobility as the protease-resistant {tau}B complex (8) generated from the endogenous yeast TFIIIC (Fig. 2D, compare lanes 3 and 5). Therefore, when bound to tDNA, the reconstituted r{tau}B and the {tau}B 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 {tau}138, {tau}91, and {tau}60.

The other DNA-binding domain of TFIIIC, {tau}A, has never been isolated. We tried to reconstitute a stable r{tau}A complex using individual recombinant polypeptides. Interestingly, co-expressed GST-{tau}131, HA-{tau}95, and {tau}55 co-purified during two successive affinity chromatographic steps (immunopurification on anti-HA-protein A-Sepharose followed by a glutathione affinity chromatography on fast protein liquid chromatography), as revealed by Coomassie Blue staining (Fig. 3A). Furthermore, the three proteins co-purified during gel filtration (data not shown). The stable complex formed by {tau}131, {tau}95, and {tau}55 likely corresponded to the postulated {tau}A module (8). We analyzed the DNA binding properties of this complex in gel shift assays using two different probes (LeuA and LeuB) harboring the A or B block of the tDNA3Leu gene, respectively. As shown in Fig. 3B, r{tau}A retarded the migration of the two DNA fragments with the same efficiency. Similar results were obtained using a nonspecific 200-bp DNA fragment as a probe (data not shown). The three subunits {tau}131, {tau}95, and {tau}55 were necessary to form a complex showing this nonspecific DNA binding activity. Indeed, none of the subunits, expressed alone or in pairs, was able to bind DNA detectably in gel shift assays under the conditions used (data not shown). To determine which subunit of {tau}A was responsible for this DNA binding activity, partially purified recombinant subunits were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and then incubated with a 32P-labeled DNA probe. As shown in Fig. 3C, only {tau}91, used as control for nonspecific DNA binding activity, and {tau}95 were able to bind DNA nonspecifically.


Figure 3
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  		FIGURE 3.
Reconstitution of r{tau}A. A, after co-expression in insect cells, GST-{tau}131, HA-{tau}95, and {tau}55 formed a stable complex that was purified successively by anti-HA protein A-Sepharose and glutathione-Sepharose affinity chromatography. The eluted proteins (0.5 µg) were analyzed with 8% SDS-PAGE and Coomassie Blue staining. The position of r{tau}A subunits is indicated. B, r{tau}A has a nonspecific DNA binding activity. Increasing amounts of purified r{tau}A (0, 20, 40, 80, and 160 ng) were assayed in gel shift, using two probes harboring the A block (LeuA) or the B block (LeuB) of the Formula gene as indicated. Protein-DNA complexes were analyzed by electrophoresis and autoradiography. C, Southwestern analysis of the DNA binding activity of r{tau}A subunits. 5 µg of partially purified recombinant {tau}131, {tau}95, {tau}55, or {tau}91 (used as a control for nonspecific DNA binding activity) were resolved on a 8% SDS-PAGE, transferred onto nitrocellulose, and then incubated with labeled poly(dA-dT) as described under "Materials and Methods." Bound DNA was revealed by autoradiography.

 
Reconstitution of Fully Recombinant rTFIIIC—To produce complete recombinant rTFIIIC, High Five cells were co-infected with six recombinant baculoviruses encoding, respectively, {tau}138, His-{tau}131, HA-{tau}95, {tau}91, {tau}60, and {tau}55. Cell extracts were loaded on a heparin column, and the protein fractions, obtained after an elution by a salt gradient, were analyzed for specific DNA binding and transcriptional activities. A control High Five cell protein extract, expressing only {tau}60 and purified under the same conditions, showed neither transcriptional nor DNA binding activities, indicating that endogenous insect TFIIIC was not purified in this way or that it was inefficient in the reconstituted yeast system (data not shown). Electrophoretic mobility shift assays on a Formula probe revealed a major protein-tDNA complex in fractions 30–50 (Fig. 4A, upper panel) that had the same mobility as the endogenous yTFIIIC-tDNA complex (Fig. 4A, compare lanes 2 and 5). A minor faster complex that co-eluted with the upper complex probably corresponded to r{tau}B. A complex migrating slightly faster than yTFIIIC-tDNA complex was also formed with fractions 50–70. Remarkably, specific transcription activity was detected in fractions containing both complexes (Fig. 4A, lower panel, lanes 5–9). These two putative forms of TFIIIC (called rTFIIICa and rTFIIICb) were further purified separately on a metal chelate affinity column by pooling separately fractions 30–40 and fractions 60–80 that do not form the upper complex. As revealed by silver staining, the recombinant complexes, rTFIIICa and rTFIIICb, eluted from the affinity column, were highly purified (Fig. 4B). Both complexes contained the six subunits of TFIIIC ({tau}138, {tau}131, {tau}95, {tau}91, {tau}60, and {tau}55). rTFIIICa contained one additional band, marked by an asterisk, which was identified by mass spectrometry as a proteolytic product of {tau}138. rTFIIICb contained two additional polypeptides that corresponded to a degradation product of {tau}131 and to the N-terminal part of {tau}95, respectively. At this stage of purification, we could not rule out the possibility that our fractions contained subcomplexes that may contaminate the complete rTFIIIC. However, the stoichiometry of each subunit was quite similar in recombinant and endogenous TFIIIC factor complexes, as revealed by Western blot analysis (data not shown).


Figure 4
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  		FIGURE 4.
Characterization of recombinant rTFIIIC. A, DNA binding and transcriptional activities of heparin-purified TFIIIC. Insect protein extracts co-expressing {tau}138, His-{tau}131, Ha-{tau}95, {tau}91, {tau}60, and {tau}55 were subjected to heparin chromatography. The eluted fractions were assayed for DNA binding activity by mobility shift assays with the Formula gene as a probe (upper panel) or for transcriptional activity with the SUP4 tRNATyr gene as a template, in the presence of the B'' fraction, rTBP, rBrf1, and purified RNA Pol III, as described under "Materials and Methods." Upper panel, lane 1, free DNA probe; lane 2, yTFIIIC; lanes 3–11, 1 µl of fractions 10–90. The positions of the rTFIIICa, rTFIIICb, and r{tau}B-DNA complexes are indicated on the right. Lower panel, lane 1, no TFIIIC; lane 2, yTFIIIC; lanes 3–11,1 µl of fractions 10–90. rTFIIICa (fractions 30–50) is eluted from 420–540 mM AS, and rTFIIICb (fractions 50–70) is eluted from 540–620 mM AS. B, polypeptide content of rTFIIICa and rTFIIICb. Highly purified rTFIIICa and rTFIIICb (10 µl of metal chelate affinity step) were analyzed by 8% SDS-PAGE and silver-stained. Molecular weight markers are indicated on the left, and TFIIIC subunits and some proteolytic by-products are indicated (*) on the right.

 
Functional Properties of rTFIIIC—In gel shift assays, rTFIIICa and rTFIIICb formed two distinct complexes (Fig. 4A). The same results were obtained with different tRNA genes with varying intron length, indicating that the difference in complex mobility was not gene-specific (data not shown). DNA complex formation with recombinant TFIIICa or TFIIICb or endogenous TFIIIC had a similar sensitivity to salt concentrations and temperature (data not shown). To compare the relative affinity to tDNA of both forms of rTFIIIC, we determined the apparent dissociation constant (Kapp) of TFIIIC-DNA complexes by titrating a fixed amount of TFIIIC (yTFIIIC, rTFIIICa, or rTFIIICb) with varying concentrations of Formula gene under optimal binding conditions (38). TFIIIC-DNA complexes were resolved by gel electrophoresis and quantified by a PhosphorImager. The linearity of the Scatchard representation (Fig. 5A) suggested the presence of a single, major binding component in the protein-DNA complexes. From three independent Formula titration experiments, the Kapp estimated for rTFIIICa, rTFIIICb, and endogenous yTFIIIC was not significantly different (1.4 x 10–11, 2 x 10–11, and 2.6 x 10–11M, respectively). The DNA binding specificity of the endogenous or recombinant factor was further compared in DNA footprinting assays using the same amount of factor titrated by gel shift assays. As shown in Fig. 5B, the footprints of yTFIIIC and rTFIIICa on the Formula gene were indistinguishable (Fig. 5B, lanes 1 and 2). In contrast, rTFIIICb yielded a comparatively weaker footprint over the A block region (Fig. 5B, lane 3). Endogenous and recombinant TFIIIC were then compared for their ability to direct specific transcription of various tRNA and 5 S RNA genes in vitro. Wondering whether the reconstituted rTFIIIC had the same flexibility as the endogenous factor, we used tRNA genes with variously spaced A and B blocks. Similar amounts of yTFIIIC (y), rTFIIICa (Ca), and rTFIIICb (Cb), based on gel shift data, were used in reconstituted transcription assays in the presence of the B'' fraction (containing Bdp1), recombinant TBP, rBrf1, highly purified RNA polymerase III, and rTFIIIA when indicated. As expected, the in vitro transcription system was TFIIIC-dependent because no transcription could be detected in the absence of the factor (Fig. 5C). Both recombinant rTFIIIC, Ca or Cb, directed the transcription of the tRNA genes assayed. No difference in the length of the transcripts could be detected when compared with the transcripts obtained with endogenous yTFIIIC (Fig. 5C compare lanes y, Ca, and Cb), suggesting that the initiation and termination processes occurred correctly. In the presence of rTFIIIA, both forms of rTFIIIC could direct transcription of the 5SRNA gene (Fig. 5C). Taking advantage of the SUP4 tRNATyr gene sequence, which allows the synthesis of a 17-mer transcript in the absence of GTP (Fig. 5D), we analyzed the specificity of the initiation reaction. Both recombinant and endogenous TFIIIC were able to synthesize the expected 17-mer RNA, indicating that recombinant rTFIIIC allowed initiation of transcription at the correct site (Fig. 5D, compare lanes 2–4).

Effect of Phosphatase Treatments on TFIIIC Activities—Three subunits of the yeast TFIIIC, {tau}131, {tau}95, and {tau}138 have been found to be phosphorylated in vivo (6, 44), whereas {tau}91 and {tau}55 have been found to be phosphorylated in vitro (45). The function of these modifications has not been established yet. In view of the presence of two chromatographic forms of rTFIIIC, we explored the effect of the dephosphorylation of rTFIIICa on DNA binding using gel shift assays. As shown in Fig. 6A, PAP treatment of partially purified rTFIIICa resulted in the formation of DNA complexes of slightly increased electrophoretic mobility, comparable with that of rTFIIICb. In contrast, when phosphatase inhibitors were added together with PAP during the preincubation period with phosphatase, rTFIIICa-DNA complex migration remained unaffected (Fig. 6A, compare lanes 1 and 7). Preincubation of rTFIIICb with PAP under the same conditions also resulted in the formation of complexes of slightly increased mobility (data not shown), suggesting that rTFIIICb could also be phosphorylated. Although PAP is not able to convert rTFIIICa to rTFIIICb, rTFIIICb could be a partially dephosphorylated form of rTFIIICa. Similar results were obtained with partially purified yeast endogenous TFIIIC (Fig. 6A, lanes 8–12) and with highly purified rTFII-ICa (Fig. 6B). We analyzed the effect of the phosphatase treatment on TFIIIC activities. Highly purified rTFIIICa was preincubated with varying amounts of PAP at 30 °C and then assayed for DNA binding and transcription factor activities. As shown in Fig. 6B, dephosphorylation treatments resulted in a marked decrease in transcriptional activity, but this decrease was directly correlated with the drop of rTFIIICa-tDNA complex formation. Phosphatase inhibitors prevented the increased mobility of the complex and markedly reduced the drop in DNA binding and transcription activities. Nevertheless, we could not rule out the fact that a part of this effect could be attributed to some TFIIIC degradation, especially when pure rTFIIIC is used in the presence of the PAP. The dephosphorylation treatment affected both the DNA binding and the transcriptional activities of TFIIIC. However, one cannot ascertain that the transcriptional defect is not simply because of the defective DNA binding.


Figure 5
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  		FIGURE 5.
Functional characterization of rTFIIIC. A, tDNA affinity of rTFIIIC. TFIIIC-DNA complexes formed with varying amounts of labeled Formula probe and equal amounts of TFIIIC (yTFIIIC, rTFIIICa, or rTFIIICb) were analyzed by gel retardation assays as described under "Materials and Methods." The Scatchard representation is shown and was used to calculate the apparent dissociation constants (Kapp) of TFIIIC-DNA complexes. B, footprint of purified endogenous yTFIIIC (lanes 1), highly purified rTFIIICa (lanes 2), or rTFIIICb (lanes 3) on the Formula gene. Lane 4, control DNA without bound protein. The diagram on the side shows the location of the A and B blocks and the start site. C, transcriptional activity of rTFIIIC. Transcriptions reactions contained different plasmid templates as indicated: purified endogenous yTFIIIC (y) or highly purified recombinant rTFIIICa or rTFIIICb (Ca and Cb, respectively), rTBP, rBrf1, B'' fraction, purified RNA Pol III, and rTFIIIA with the 5S RNA template. RNA transcripts were analyzed by electrophoresis and autoradiography. D, analysis of transcriptional initiation. Stable preinitiation complexes were formed on the SUP4 tRNA gene with rTBP, rBrf1, B'' fraction, and highly purified rTFIIICa or rTFIIICb or endogenous yTFIIIC. RNA Pol III was then added, and transcription was allowed to proceed for 10 min at 25 °C in the absence of GTP. The reaction products were analyzed by electrophoresis and autoradiography. The 17-mer transcript obtained in the absence of GTP is shown (right panel). Lane 1, no TFIIIC.

 
Reconstitution of a Basal RNA Polymerase III Transcription System— Next we attempted to reconstitute a defined RNA polymerase III transcription system using recombinant factors, rTBP, rBrf1, rBdp1, rTFIIIC, and highly purified RNA polymerase III. Recombinant TFIIIC and the recombinant components of yeast TFIIIB were necessary and sufficient for accurate transcription of the SUP4 tRNA gene (Fig. 7). Purified rTFIIIA was the only additional factor necessary to transcribe the 5 S RNA gene in our reconstituted system (Fig. 7). However, in each case, the level of transcription was low when rBdp1 was used instead of the B'' fraction (Fig. 7, compare lanes 1 and 2). We estimated that the transcription level was stimulated 6-fold when the B'' fraction replaced rBdp1 (Fig. 7). Similar results were previously obtained with yTFIIIC, suggesting that the B'' fraction might contain some transcriptionally active components in addition to Bdp1 (31, 46). The fact that the 5SRNA transcripts obtained with B'' were processed, whereas only precursor of 5 S RNA was obtained with rBdp1, indicated that additional activities were present in the B'' fraction (Fig. 7, lanes 9 and 10).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we showed that active yeast TFIIIC can be reconstituted from its six known recombinant subunits. The two DNA-binding domains, {tau}A and {tau}B, were characterized and could be assembled independently. Our results define a minimal transcription system, comprising all the general class III transcription factors, which could be stimulated by some unidentified components present in the B'' fraction, underscoring the need for additional auxiliary factors.

With the reconstitution of active TFIIIC from recombinant subunits, the characterization of the polypeptide composition of the yeast factor is now completed. The six subunits {tau}138, {tau}131, {tau}95, {tau}91, {tau}60, and {tau}55 are necessary and sufficient to form active rTFIIIC, which was functionally indistinguishable from endogenous TFIIIC in terms of DNA binding specificity and affinity as well as transcription factor activity. Yeast (S. cerevisiae) TFIIIC likely corresponds to human TFIIIC2, although the later comprises only five subunits and apparently lacks the ortholog of {tau}55 (47–49). Interestingly, the most conserved subunits ({tau}131 and {tau}95) are involved in A block binding and TFIIIB recruitment. The highly divergent subunits are located at the downstream end of the genes (11, 15).


Figure 6
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  		FIGURE 6.
Effect of phosphatase treatment on TFIIIC activities. A, dephosphorylation of TFIIIC and DNA binding activity. Heparin-purified rTFIIICa (lanes 1–7) or Mono Q-purified endogenous yTFIIIC (lanes 8–12) were preincubated during 30 min at 30 °C with varying amounts of PAP before DNA complex formation on a labeled Formula probe. Protein-DNA complexes were analyzed by electrophoresis and autoradiography. Lanes 1 and 8, no PAP; lanes 2–5, 0.05, 0.15, 0.25, or 0.5 unit of PAP, respectively; lane 6, rTFIIICb, no PAP; lane 7, 0.5 unit of PAP in the presence of Phosphatase inhibitor mixture; lanes 9–11, 0.25, 0.5, or 0.75 unit of PAP, respectively; lane 12, 0.75 unit of PAP in the presence of phosphatase inhibitor mixture. The migration of native yTFIIIC, rTFIIICa, rTFIIICb, and r{tau}B-tDNA complexes are indicated. B, dephosphorylation of TFIIIC and transcriptional activity. Highly purified rTFIIICa was preincubated with 0, 0.025, 0.075, 0.22, or 0.41 unit of PAP (lanes 1–5, respectively) or with 0.41 unit of PAP in the presence of phosphatase inhibitor mixture (lane 6). Phosphatase inhibitor mixture was then added, and TFIIIC activity was tested on SUP4 tRNA gene by gel shift (upper panel) or by transcription assays (lower panel). TFIIIC-DNA complexes or RNA transcripts were analyzed by electrophoresis and autoradiography. DNA complex formation and transcriptional activity were then plotted as percentages of control activity in the absence of PAP.

 


Figure 7
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  		FIGURE 7.
A minimal TFIIIC-dependent Pol III transcription system. In vitro transcription of the SUP4 tRNA (lanes 1–7) and 5 S RNA (lanes 8–10) gene was carried out in the presence of the entire set of recombinant components, rTBP, rBrf1, rBdp1, rTFII-ICa, and purified RNA Pol III (lane 2), in the presence of rTFIIIA (lane 9) or in partially reconstituted systems lacking one component as indicated. In lanes 1 and 10, the B'' fraction was used instead of rBdp1.

 
We present evidence that modification of endogenous or recombinant TFIIIC by phosphorylation is important for its DNA binding activity. TFIIIC is phosphorylated in yeast on several subunits (6, 44). Whether the same sites are phosphorylated in recombinant TFIIIC remains to be determined. Nevertheless, our observations suggest a structural and potentially regulatory role for TFIIIC modification by phosphorylation.

The present work establishes the organization of the six subunits of TFIIIC in two large globular domains, {tau}A and {tau}B (8, 9), that can be assembled independently. r{tau}B was reconstituted with {tau}138, {tau}91, and {tau}60. r{tau}B displayed the expected DNA binding specificity for the B block region and had similar affinity for the tRNA3Glu gene as TFIIIC (data not shown), confirming that {tau}B is mostly responsible for the overall stability of the TFIIIC-DNA complex (50). Interestingly r{tau}B appears to have protease sensitive extension(s) that can be removed without affecting DNA binding and that might possibly participate in {tau}B-{tau}A interaction. {tau}91 and {tau}60 subunits form a dimer that has been crystallized recently (14). The structure of this subcomplex has been solved5 and will complement the characterization of {tau}B. The {tau}A Domain was reconstituted with {tau}131, {tau}95, and {tau}55 to give a stable protein complex that displayed nonspecific DNA binding activity, which could be, at least in part, attributed to {tau}95 subunit. The basis for the DNA sequence specificity of {tau}A within TFIIIC remains intriguing. Free {tau}A may need to interact with {tau}B to form a properly folded DNA-binding domain. More specifically, as previously suggested by scanning electron microscopy analyses of TFIIIC-DNA complexes, the binding of {tau}B to the B block may induce a conformation change that allows {tau}A to recognize its target sequence (9). Note that, when preassembled independently, r{tau}A and r{tau}B did not interact readily in vitro, and we failed to reconstitute TFIIIC activity from the two subassemblies.

Well defined, minimal systems with recombinant factors have been previously described for transcription of TFIIIC-independent templates from yeast and human cells. Transcription of the human U6 RNA gene could be reconstituted with recombinant SNAPc (five subunits), TBP, Brf2, and Bdp1 (52). CK2 activity, present in the purified RNA polymerase fraction, was found to be required for transcription in this reconstituted system (53). Transcription of the yeast U6 RNA gene, that has a strong TATA box allowing proper TFIIIB assembly in vitro through TBP binding, was simply reconstituted with the recombinant components of yeast TFIIIB, TBP, Brf1, and Bdp1 and purified RNA Pol III (31, 32, 54). Remarkably, in our hands, this basal system was suboptimal and was strongly stimulated by TFIIIE, a factor that remains to be characterized (31, 35). Suboptimal transcription with entirely recombinant TFIIIB was ascribed to a defective reinitiation rate (37). Transcription reinitiation strongly influences the level of transcription, which is well described for the Pol III system (55). These observations suggested that some important component or catalytic activity was missing. Phosphorylation of TBP (56) and of the Pol III complex (53) by CK2 is required for transcription. However, the fact that the yeast CK2 can be replaced by its Drosophila ortholog in yeast cells (57) suggested that recombinant TBP was correctly modified in insect cells. A number of accessory factors have been proposed to stimulate Pol III transcription, without apparent gene specificity, including TFIIIE, the La protein, NF1, PC4, and the Topo I (reviewed in Ref. 51). The high mobility group Nhp6 protein specifically increases the transcription efficiency of SNR6 gene in vivo and in vitro by facilitating transcription complex assembly (33, 34). The present development of a reconstituted Pol III transcription system with all of the recombinant factors opens new avenues for the structural and functional analysis of TFIIIC and for the exploration of accessory effectors of the Pol III transcription system.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: CMU/Département de Microbiologie et de Médecine Moléculaire,Genève, Suisse. Back

2 Present address: Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD 20892-4255. Back

3 To whom correspondence should be addressed. Tel.: 33-1-69083796; Fax: 33-1-69084712; E-mail: joel.acker{at}cea.fr.

4 The 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. Back

5 A. Mylona and C. W. Muller, personnal communication. Back


   ACKNOWLEDGMENTS
 
We thank G. Dieci for the generous gift of purified rTFIIIA, E. Favry for protein preparations, and Fouad Ali for excellent technical assistance. Special thanks to M. Court and Jerome Garin (LCP, CEA/Grenoble) for the mass spectrometry analysis. We are grateful to S. Camier, C. Conesa, S. Jourdain, and Christian Marck for helpful discussion. We thank Nazif Alic for improving the manuscript.



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