INTRODUCTION

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The primary step in tRNA gene activation is the binding of TFIIIC to the A and B blocks of the intragenic promoter. Its main function is then to assemble the initiation factor TFIIIB upstream of the transcription start site (30). Yeast TFIIIC ( factor) is a multisubunit protein of ca. 600 kDa organized in two large globular domains A and B of similar size and mass (10-nm diameter and ca. 300 kDa), each interacting with one promoter element, as visualized by electron microscopy (17, 53). Binding of B to the B block is predominant and favors the binding of A to the A block (4). TFIIIC-DNA interaction displays a remarkable adaptability to the variable A-B distances found in different tRNA genes (3). Affinity-purified Saccharomyces cerevisiae TFIIIC comprises six polypeptides of 138, 131, 95, 91, 60, and 55 kDa (6, 19, 47, 59). Gene cloning, protein-DNA cross-linking, mutagenesis, and protein-protein interaction studies provided a global view of the location and role of several TFIIIC subunits within the TFIIIC-DNA complex. 138 and 91 subunits reside in the B domain and cooperate in downstream DNA binding (1, 10, 36, 37); 95 and 55 interact physically, belong to the A domain, and are thought to participate in A block binding (7, 17, 40, 48, 59). 131 (42) stands as the TFIIIC subunit responsible for TFIIIB assembly based on genetic evidence (49, 61), its upstream location (7), and its interaction with two TFIIIB components (14, 33, 51).

TFIIIB is not a stable molecular entity like TFIIIC. It can be resolved chromatographically into two fractions named B' and B" (29). B' comprises TATA-binding protein (TBP) and the TFIIB-related factor TFIIIB70/Brf1 (12, 16, 31, 39), while B" contains TFIIIB90 (32, 50, 51). The TFIIIC-dependent TFIIIB assembly on TATA-less class III genes is a multistep pathway that could be decomposed in vitro (29, 31) and reconstituted with recombinant TFIIIB components (32, 51). The order of interaction is TFIIIB70, then TBP, and then B", as shown by gel retardation and DNA photo-cross-linking (31). TBP stabilizes the weak interaction between TFIIIB70 and the TFIIIC-DNA complex but the complete upstream footprint and the characteristic stability of the TFIIIB-DNA complex requires the recruitment of B"/TFIIIB90 (29, 31). A cascade of conformational rearrangements at the protein and DNA levels are accompanying these assembly steps, as evidenced by successive changes in the accessibility of TFIIIB70, TBP, and 131 to site-specific DNA cross-linking (31), by the DNA bending induced upon TFIIIB binding (11, 38, 46), and by the presence of a cryptic DNA binding domain in TFIIIB70 (24). 131 appears to play the major role in positioning TFIIIB since it is the only TFIIIC subunit accessible to DNA cross-linking upstream of the start site (5, 7) and found to interact with TFIIIB70 (14, 33) and TFIIIB90 (51). TFIIIB can effect its own assembly onto the TATA-containing SNR6 gene through the interaction of TBP with the strong TATA box (27, 43, 45). Interestingly, Whitehall et al. (60) found that TBP could not discern the polarity of the TATA element and directed TFIIIB assembly in two orientations. However, in contrast to the TATA-dependent assembly, TFIIIC placed TFIIIB in the correct orientation. Since no TFIIIC component was known to interact with TBP, it was presumed that the unidirectional binding of TBP to the TATA box is dictated by the oriented interaction of TFIIIB70 with 131 (60).

In the present work we have completed the characterization of TFIIIC components by cloning a yeast gene, named TFC8, that encodes the 60-kDa polypeptide. We present genetic and biochemical evidence that this component, named 60, resides at least in part within the B domain and participates in TFIIIB recruitment via TBP binding.

	MATERIALS AND METHODS

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Yeast strains, plasmids, and genetic techniques. YNN281×YNN282 (21) was used for gene disruption. Cells were grown in standard rich medium (YPD) or minimal medium (Casamino acid medium). The plasmids used for suppression studies are described in Lefebvre et al. (37). Preparation of media, tetrad dissection, transformation of lithium acetate-treated cells, and plasmid shuffling by using 5-fluoro-orotic acid were performed by standard techniques (2).

Amino acid sequence determination. The polypeptide components of purified TFIIIC preparation were separated by preparative electrophoresis on a 6% polyacrylamide-sodium dodecyl sulfate (SDS) gel (36). The gel was lightly stained with Coomassie blue. A gel slice containing the 60-kDa polypeptide was excised, crushed, and subjected to trypsin digestion. Preparation and analysis of tryptic peptides have been previously described (36, 59). One peptide sequence was partially determined (??TLYLTT[T/F]PT).

Cloning, construction of plasmids, and disruption of TFC8. Two 32-mer oligonucleotides were used to amplify the open reading (ORF) frame of TFC8 by PCR on yeast genomic DNA. The resulting DNA fragment was then labeled with [-³²P]dCTP and used to screen the FL100 library (57) containing yeast genomic DNA fragments inserted into the pFL44L (2µ, URA3) plasmid (9). The pFL44L plasmid isolated from one of the hybridizing clones was found to contain a large DNA insert comprising the TFC8 gene and was named pCC12 (pFL44L-TFC8). The 2.3-kb SalI-FspI DNA fragment from pCC12 harboring the TFC8 gene was cloned into pUN45 creating the pYED1 plasmid. A 69-mer oligonucleotide was used to introduce a NdeI restriction site at the initiation codon of TFC8, followed by the sequence encoding the hemagglutinin (HA) epitope (YPYDVPDYA) derived from the influenza virus HA protein (62), by oligonucleotide-mediated mutagenesis on single-stranded pYED1 DNA by using a Muta-Gene kit (Bio-Rad), yielding the pYED2 plasmid (encoding HA Nter-60). The sequence encoding the HA epitope was also introduced before the stop codon of TFC8 by PCR-mediated mutagenesis with two oligonucleotides. One contained a SalI restriction site and nucleotides complementary to the upstream region of the TFC8 promoter, and the other harbored the sequence encoding the HA epitope, a NotI restriction site, and nucleotides complementary to the stop codon region of TFC8. After PCR amplification, the SalI-NotI DNA fragment was inserted into the corresponding sites of pYED1 to give pYED3 (encoding for HA Cter-60). The NdeI-BamHI DNA fragment from pYED2 was inserted into the corresponding sites of pET28c vector (Novagen), yielding pET60.

Purification of TFIIIC. TFIIIC was purified starting from ~12 g of S. cerevisiae cells, using fast-protein liquid chromatography-grade resins. The preparation of the cell extract was done as described by Huet et al. (25). Crude extract was first diluted to 0.25 M ammonium sulfate (AS) with buffer I (20 mM Tris-HCl, pH 8.0; 0.5 mM EDTA; 10 mM -mercaptoethanol; 10% [vol/vol] glycerol) and then loaded at 2.5 ml/min on a 25-ml heparin Hyper D (BioSepra) column previously equilibrated with buffer I (0.25 M AS). The resin was then washed at 5 ml/min with 250 ml of buffer I (0.35 M AS). A linear gradient of AS from 0.35 to 0.70 M in 180 ml of buffer I was then applied at 2.5 ml/min. Fractions (2 ml) were collected and assayed for TFIIIC-DNA binding activity. TFIIIC-containing fractions (0.45 to 0.55 M AS) were pooled and dialyzed against buffer I (0.07 M AS). Proteins were then loaded at 0.5 ml/min on a 1-ml MonoQ column (Pharmacia, Piscataway, N.J.) previously equilibrated with buffer I (0.07 M AS). The column was washed at 0.5 ml/min with 20 ml of buffer I (0.07 M AS). A linear gradient of AS from 0.07 to 0.4 M in 15 ml of buffer I was then applied at 0.5 ml/min. Fractions (200 µl) containing TFIIIC-DNA binding activity were eluted between 0.24 and 0.30 M AS. Based on Western blot experiments with anti-55 or anti-60 antibodies, the TFIIIC preparation from Ntag-60 mutant cells was found to contain half as much factor as the wild type.

Expression and purification of 60 in Escherichia coli. Recombinant Tfc8p tagged at its N-terminal end with six histidines and with the HA epitope (HA-r60) was expressed from plasmid pET60 in E. coli BL21 (pLysS). Cell culture, protein induction, and extract preparation were performed as described earlier (1). Crude cell extract was loaded at 1 ml/min on a 5 ml of Ni²⁺-charged HiTrap chelating column (Pharmacia) previously equilibrated with 20 mM HEPES (pH 7.8), 300 mM NaCl, and 10% glycerol containing 10 mM imidazole. Proteins were eluted by a linear gradient in 60 ml of the same buffer containing 20 to 270 mM imidazole at 1 ml/min. Fractions (1 ml) were assayed for HA-r60 by Western blotting with anti-60 antibodies. Fractions containing HA-r60 were eluted at concentrations between 160 and 190 mM imidazole.

Anti-60 polyclonal antibodies. A 14-mer peptide (N-12-M) encompassing the C-terminal amino acid residues of 60 was synthesized (Neosystem, Strasbourg, France) and conjugated to maleimide-activated keyhole limpet hemocyanin (KLH). N-12-M-KLH conjugate was then injected into rabbits for antibody production in three injections at about three week intervals.

Immunoprecipitation experiments. rTBP alone (200 ng) or both rTBP (200 ng) and HA-r60 (650 ng) were preincubated for 90 min at 25°C in 40 µl of buffer A containing 20 mM HEPES (pH 8.0), 5 mM MgCl₂, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 5% glycerol, 0.05% NP-40, and 110 mM KCl. Magnetic beads (Dynabeads/Dynal) coated with 12CA5 antibody were added to the mixtures. The beads were incubated with gentle shaking for 3 h at 10°C and then washed three times in buffer A. Bound proteins were eluted by boiling in Laemmli sample buffer, separated by SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blotting by using a mixture of polyclonal antibodies directed to TBP and to 60. Bound antibodies were revealed by using the Amersham ECL Kit. The polypeptides revealed by antibodies directed to TBP or 60 were identified in independent experiments.

DNA binding and in vitro transcription assays. TFIIIC-tDNA or B-tDNA interaction was monitored by gel retardation analysis as described previously (25, 37). A ³²P-labeled DNA fragment (3 to 10 fmol; 4,000 to 10,000 cpm) carrying the tRNA₃^Glu (198 bp) or the SUP4tRNA^Tyr (375 bp) genes was incubated for 10 min at 25°C in a 15-µl reaction mixture containing 10 mM Tris-HCl (pH 8.0), 10% glycerol, bovine serum albumin, DNA competitor, and TFIIIC (MonoQ fraction). The final ammonium sulfate concentration was 75 mM. B-tDNA complexes were obtained after a further 10 min incubation at 25°C of TFIIIC-tDNA complexes with 10 ng of -chymotrypsin (Sigma). Digestion was stopped by addition of 1 ng of aprotinin (Sigma). Complexes were analyzed by nondenaturing gel electrophoresis (5% polyacrylamide).

In vivo labeling of RNAs and Northern blot analysis. RNA labeling was done with [³H]uracil with strains where the ura3 mutation was complemented by the URA3 plasmid pFL44L. Cells were exponentially grown in uracil-free Casamino acid medium supplemented with adenine (Casa+Ade) to an optical density of 0.4 at 600 nm. Then, 150 µCi of [³H]uracil was added to 10 ml of culture for 15 min. The cells were next harvested and chilled with 10 ml of ice-cold sterile water. RNAs were extracted as previously described (22, 52). Small RNA species were analyzed by loading and separating equal amounts of RNA (6 µg per lane) on a 7 M urea gel by electrophoresis (6% polyacrylamide).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

TFC8 encodes 60, the 60-kDa subunit of yeast TFIIIC. TFIIIC from S. cerevisiae comprises six polypeptides of 138, 131, 95, 91, 60, and 55 kDa. In order to clone the gene encoding the hypothetical 60 subunit, the polypeptide was purified by SDS-PAGE and subjected to tryptic digestion. The sequence of only one peptide could be determined (??TLYLTT[T/F]PT). When compared to DNA sequences in databases by using the BLAST program, this peptide sequence was found with a slight variation (DGTLYLTTFPD) in a unique yeast hypothetical protein of 588 residues, with a theoretical molecular weight of 67,640 and an isoelectric point of 5.87. The gene encoding this protein is unique, maps on chromosome XVI (13), and shows no similarity to any sequences in the EMBL/GenBank data bank (S. cerevisiae, Caenorhabditis elegans, and current versions of Arabidopsis thaliana, Homo sapiens, and Schizosaccharomyces pombe genomic sequences). The gene, previously designated YPL007C, was named TFC8.

View larger version (54K): [in this window] [in a new window]   FIG. 1.   TFC8 encodes the 60-kDa subunit of yeast TFIIIC. (A) TFIIIC was purified from haploid strains expressing HA-tagged versions of TFC3 (HA-138) or TFC8 (Ctag-60 or Ntag-60, tagged at the C-terminal or N-terminal end of Tfc8p, respectively). TFIIIC was preincubated with a 32P-labeled DNA fragment containing the tRNA3Glu gene for 10 min at 25°C and then further incubated with various amounts of 12CA5 monoclonal antibody directed to the HA epitope for 30 min at 25°C. Protein-tDNA complexes were analyzed by gel retardation assay and revealed by autoradiography. Lanes 1 and 8, no TFIIIC, no antibody; lanes 2, 7, 9, or 11, no antibody; lanes 3 to 6, 10, and 12, addition of 12CA5 monoclonal antibody, as indicated. (B) B, the protease-resistant domain of TFIIIC, HA-tagged on 138 or 60, was obtained after digestion of the TFIIIC-tDNA complex with 10 ng of -chymotrypsin for 10 min at 25°C. Digestion was stopped with 1 ng of aprotinin before addition of the 12CA5 antibody. Left panel, C-terminally HA-tagged TFC8 gene (Ctag-60); right panel, HA-tagged TFC3 gene (HA-138) or N-terminally HA-tagged TFC8 gene (Ntag-60). Lanes 1 and 9, control TFIIIC-tDNA complex; lanes 2, 7, and 10, B generated by proteolysis; lanes 3 to 6, 8, and 11, incubation of B-tDNA complex with various amounts of 12CA5 monoclonal antibody, as indicated. , TFIIIC-tDNA complex; -IgG, immunoglobulin G (IgG)-TFIIIC-tDNA ternary complex; B, B-tDNA complex; B-IgG, IgG-B-tDNA ternary complex.

In vivo characterization of the Ntag-60 mutant. We took advantage of the thermosensitive phenotype of the Ntag-60 mutant to explore the role of the TFC8 gene product in TFIIIC. The effect of the mutation on Pol III transcripts in vivo is shown in Fig. 2A. The wild-type and mutant cells were grown at 30°C and then shifted or not to nonpermissive temperature (37°C) for 6 or 12 h. The cells grown at 30 or 37°C were incubated at the same temperature with tritiated uracil for 15 min; the RNAs were then extracted, separated on a 7 M urea gel, and analyzed by autoradiography. No difference was observed in the RNA labeling pattern of the wild-type and mutant cells grown at 30°C (lanes 1 and 2). In contrast, when shifted for 6 or 12 h at 37°C, the mutation altered the labeling pattern of tRNAs. A smear of radioactivity was detected just above the labeled tRNA bands (lanes 4 and 5), which could correspond to partially matured tRNAs, and after 12 h the labeling of the tRNA bands was selectively reduced (lane 5). Clearly, the fusion of the HA epitope to 60 at the N-terminal extremity disturbed the Pol III transcription system. Like in previous studies on different Pol III system mutants, the synthesis of 5S RNA did not decrease significantly (20, 41).

View larger version (38K): [in this window] [in a new window]   FIG. 2.   Comparison of in vivo tRNA synthesis in wild-type and mutant (Ntag-60) yeast strains. (A) Autoradiogram of the in vivo pulse-labeling experiments. [3H]uracil incorporation into wild-type and mutant strains was done for 15 min at 30°C (lane 1, wild-type; lane 2, mutant) or at 37°C after 6 h (lane 4, mutant) or 12 h (lane 3, wild-type; lane 5, mutant) of growth at 37°C. RNA species were analyzed by electrophoresis on a 7 M urea gel (6% polyacrylamide) by using equal amounts of RNA (6 µg per lane). The asterisk indicates the trailing of radioactive material above the tRNA species, suggesting a defect in tRNA maturation. (B) Northern blot analysis of tRNA precursors from wild-type and mutant cells with a labeled tDNA3Leu probe. RNA extraction was done after 8 h of growth at 37°C. Gel electrophoresis, electrophoretic transfer on nylon membrane, and hybridization with DNA probe was done as described in Materials and Methods. The positions of the primary transcript of tRNA3Leu, the 5'- and 3'-processed but nonspliced precursor, the spliced but 5'- and 3'-unprocessed precursor, and the mature tRNA3Leu are indicated on the right. Lanes 1 and 2, wild-type and mutant cells, respectively, containing the high-copy-number plasmid pFL44L; lane 3, mutant cells containing the high-copy-number plasmid pFL44L harboring RPR1 gene; lane 4, mutant cells containing pFL44L harboring SPT15 gene (TBP).

View larger version (57K): [in this window] [in a new window]   FIG. 3.   Suppressibility of Ntag-60 mutation by overexpression of Pol III-related genes. Stationary-phase cultures of mutant cells harboring different multicopy plasmids were diluted 10-, 102-, and 103-fold in water and spotted (5 µl) onto solid minimum medium (Casa+Ade plates). The plates were incubated for 4 days at 37°C. The different genes harbored on the high-copy-number vector pFL44L are indicated. , plasmid without insert. RPR1 encodes the RNA cofactor of RNase P.

View this table: [in this window] [in a new window]   TABLE 1.   Suppression of 60 mutation by gene dosage

In vitro characterization of the Ntag-60 mutant. TFIIIC factor was partially purified from a wild-type strain and from the strain expressing the HA N-terminal version of 60 to compare their DNA binding properties. TFIIIC fractions purified from the mutant strain were reproducibly found by Western blotting with anti-60 and anti-55 polyclonal antibodies (40) to contain half as much TFIIIC as wild-type fractions. Therefore, care was taken to use the same amount of wild-type or mutant TFIIIC in the following gel retardation experiments. Two labeled probes, tDNA₃^Glu or SUP4tDNA^Tyr, were incubated with limiting amounts of wild-type or mutant TFIIIC. TFIIIC-tDNA complexes were analyzed by electrophoresis under nondenaturing conditions and revealed by autoradiography. Figure 4A shows that the yields of TFIIIC-DNA complexes formed with wild-type or mutant TFIIIC factor on tDNA₃^Glu or SUP4tDNA^Tyr were very similar. In addition, wild-type and mutant TFIIIC-tDNA complex stabilities at various temperatures or ionic strengths were also indistinguishable (data not shown). In contrast, when TFIIIC-tDNA₃^Glu complexes were subjected to limited proteolysis, significantly fewer mutant B-tDNA complexes were obtained compared to the wild type (Fig. 4B, compare lanes 2 and 9). Moreover, mutant B-tDNA complexes were less stable than wild type at increasing temperatures. The amount of mutant B-tDNA complexes decreased drastically after 10 min at 55°C to 5% of the initial control level at 25°C, while 60% of wild-type B-tDNA complexes resisted under the same conditions (lanes 6 and 13), as determined by using a PhosphorImager with ImageQuant software (Molecular Dynamics). Whatever the structural defect causing the increased thermolability (either the presence of the HA epitope or some subtle difference in proteolysis indirectly caused by the presence of the epitope), these observations clearly revealed a defect at the B level, enforcing the idea that the N-terminal part of 60 belongs to the B block-binding domain.

View larger version (72K): [in this window] [in a new window]   FIG. 4.   DNA-binding properties of wild-type and mutant TFIIIC. (A) TFIIIC-tDNA complex formation on tRNA3Glu or SUP4tRNATyr genes. TFIIIC-DNA complexes were formed with Ntag-60 or wild-type TFIIIC preparations (MonoQ fraction) and analyzed by electrophoresis as described in Materials and Methods. To add similar amounts of wild-type and mutant TFIIIC (based on Western blot experiments), twice as much mutant protein fraction was added as follows: 25 ng (lanes 2 and 7) and 100 ng (lanes 3 and 8) of wild-type TFIIIC and 50 ng (lanes 4 and 9) and 200 ng (lanes 5 and 10) of mutant TFIIIC fraction. Lanes 1 to 5, tRNA3Glu gene; lanes 6 to 10, SUP4tRNATyr gene. (B) Effect of temperature on B-tDNA complex stability. Preformed TFIIIC-tDNA3Glu complexes were digested with -chymotrypsin to generate B-DNA complexes, proteolytic digestion was stopped by addition of aprotinin, and the mixtures were then further incubated for 10 min at various temperatures as indicated. Lanes 1 and 8, wild-type and mutant TFIIIC-tDNA complexes, respectively; lanes 2 to 7, wild-type B; lanes 9 to 14, mutant B. , TFIIIC-tDNA complex; B, B-tDNA complex.

View larger version (64K): [in this window] [in a new window]   FIG. 5.   Specific transcription of various tRNA genes in the presence of wild-type or mutant TFIIIC. In vitro transcription was performed as described in Materials and Methods by using 100 and 200 ng of wild-type and Ntag-60 MonoQ-TFIIIC fraction (respectively), rTBP, rTFIIIB70, partially purified B" fraction, and RNA Pol III. The different plasmid templates are indicated. Odd lanes, wild-type TFIIIC. Even lanes, mutant TFIIIC.

View larger version (44K): [in this window] [in a new window]   FIG. 6.   Effects of TFIIIB, TFIIIB70, B", or TBP concentration on the transcription of tRNA3Leu or tRNA3Glu genes in the presence of wild-type or mutant TFIIIC. (A) Effect of TFIIIB. Transcription mixtures (40 µl) contained 100 ng of wild-type or 200 ng of Ntag-60 TFIIIC, 220 ng of plasmid DNA harboring the tRNA3Leu gene, RNA polymerase, and various concentrations of TFIIIB (Cibacron blue fraction) as indicated. (B) Effect of TFIIIB70. Transcription mixtures contained wild-type or mutant TFIIIC, plasmid DNA, and RNA polymerase as in panel A, 2.5 ng of rTBP per µl, 45 ng of partially purified B" fraction per µl, and various concentrations of rTFIIIB70 as indicated. (C) Effect of B". Transcription mixtures contained wild-type or mutant TFIIIC, plasmid DNA, and RNA polymerase as in panel A, 4 ng of rTBP per µl, 35 ng of rTFIIIB70 per µl, and various concentrations of partially purified B" fraction as indicated. (D) Effect of TBP. Lanes were as in other panels, with 35 ng of rTFIIIB70 per µl, 45 ng of partially purified B" fraction per µl, and various concentrations of rTBP as indicated. (E) Effect of TBP. Lanes are as described for panel D, except that plasmid DNA harboring the tRNA3Glu gene instead of the tRNA3Leu gene was used as the template. Transcripts were analyzed by electrophoresis and autoradiography (left panels). Odd lanes, wild-type TFIIIC; even lanes, mutant TFIIIC. Transcripts were quantified (right panels) by using a PhosphorImager and ImageQuant software (Molecular Dynamics). a.u., arbitrary units. Gray bars, wild-type TFIIIC; black bars, mutant TFIIIC. The relative transcription efficiencies of the mutant versus the wild-type TFIIIC are indicated above the bar graphs. ND, not determined. Two or three independent experiments were compiled for quantification.

60 interacts with TBP. Two lines of evidence indicated that mutant TFIIIC was defective in TBP recruitment, both in vivo (the multicopy suppressor studies) and in vitro (the transcription experiments). We therefore explored the possibility that 60 subunit might directly participate in TBP binding. The physical interaction between 60 and TBP was investigated by coimmunoprecipitation experiments with recombinant proteins. rTBP (200 ng) was preincubated for 90 min at 25°C with HA-r60 (650 ng) in transcription buffer. The protein samples were then added to magnetic beads precoated with anti-HA antibodies, the beads were washed with the same buffer, and bound proteins were eluted with SDS or by competition with the HA peptide. The input and eluted proteins were subsequently analyzed by SDS-PAGE and immunoblotting with anti-TBP and anti-60 antibodies (Fig. 7). Compared to the low background level of TBP retained by the beads in the absence of 60, a sevenfold-higher amount of TBP was bound upon preincubation with 60, as quantified by using the ImageQuant software. A similar result was obtained when bound proteins were eluted by competition with the HA peptide or when the beads were washed extensively with the transcription buffer (data not shown). The background-corrected TBP/Tfc8 ratio in the eluted fraction has been quantified and corresponded to 0.56 (compared to 0.9 in the input), showing that each molecule of bound 60 was specifically associated to 0.5 molecule of TBP. From that result, we tentatively estimate the K_d of TBP-60 interaction to be ca. 100 nM, which is indicative of a reasonably strong interaction. The same K_d was reported for TFIIIB70-TBP interaction by Sethy-Coraci et al. (55). These observations strongly suggested a direct interaction between the 60 subunit and TBP. In a similar experiment, no physical interaction could be detected between r60 and rTFIIIB70: Western blot analysis revealed no significant difference in the amounts of rTFIIIB70 bound to the beads in the presence or absence of HA-r60 (data not shown).

View larger version (90K): [in this window] [in a new window]   FIG. 7.   Coimmunoprecipitation experiment. HA-tagged r60 (650 ng) was preincubated with rTBP (200 ng) for 1.5 h at 25°C and purified on magnetic beads coated with anti-HA antibodies. Bound proteins were eluted and analyzed by Western blotting with anti-60 and anti-TBP polyclonal antibodies. The positions of rTBP and of native HA-r60 are shown. The intermediate bands are specifically revealed by anti-60 antibodies. Lane 1, rTBP+HA-r60; lane 2, rTBP alone; lane 3, 25% of input rTBP+HA-r60 proteins.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We report here the characterization of the 60-kDa polypeptide present in affinity-purified TFIIIC. This polypeptide, named 60, is shown to be an essential subunit of TFIIIC. The yeast TFIIIC factor, therefore, comprises six subunits, 138, 131, 95, 91, 60, and 55, all of which are essential for cell viability (Table 2). 60 appears to reside, at least in part, in B, the downstream DNA-binding domain. Paradoxically, however, analysis of a 60 mutant indicated that 60 plays a role in TFIIIB assembly via its interaction with TBP. The results suggest a network of interactions linking TFIIIC to TFIIIB components during preinitiation complex formation.

The demonstration that 60 is an integral subunit of TFIIIC rests on biochemical and genetic evidence. First, antibodies directed to C-terminally HA-tagged 60 altered the migration of tagged TFIIIC-tDNA and B-tDNA complexes. Second, a thermosensitive mutant TFIIIC (harboring N-terminally HA-tagged 60) was affected in tRNA gene transcription in vivo and in vitro. Third, this in vivo phenotype was suppressed by overexpression of components of the class III transcription machinery (a TFIIIC subunit, 95, and two components of the TFIIIB factor, TFIIIB90/B" and TBP). Fourth, the in vitro transcription defect was strongly alleviated by increasing the concentration of TFIIIB or TBP. Finally, like all of the subunits of TFIIIC previously characterized, the TFC8 gene encoding 60 is essential for cell viability. Altogether, these results indicate that 60 plays an important role in TFIIIC.

The location of TFIIIC subunits by site-specific DNA cross-linking has provided a broad view of the possible functions of the different polypeptides within the TFIIIC-DNA complex (Table 2), which are always in good agreement with other biochemical or genetic data (28). Unfortunately, 60 was the only TFIIIC polypeptide that could not be cross-linked and located by this elegant and powerful approach (its subunit status could have been questioned for that reason). Two independent experiments suggested that 60 resides within B domain: B-DNA complexes obtained by proteolysis of TFIIIC harboring HA-tagged 60 were found to be more thermosensitive than untagged complexes or were recognized by antibodies directed to the HA epitope. Since, in these experiments, the HA tag was placed either at the N-terminal or at the C-terminal end of the polypeptide, we inferred that both extremities of 60 resided in B, together with 138 (19) and 91 (1). The total mass of these three polypeptides, 275 kDa, would correspond reasonably well to the mass of B (~300 kDa) visualized by scanning transmission electron microscopy over the B block (53). Note that the definition of B by electron microscopy and by limited proteolysis are not necessarily equivalent. The protease-resistant form of B observed by gel retardation assay may contain pieces of TFIIIC unrelated to the B-block-binding function. The participation of 60 to B (as defined by limited proteolysis and gel retardation assay) points to a role of this subunit in DNA binding. Indeed, the presence of the HA epitope at the N-terminal end of 60 decreased the yield and weakened the stability of B-DNA complex. However, mutant TFIIIC-DNA complex formation was not notably affected compared to the wild-type factor, even under unfavorable conditions, at high ionic strengths, or at high temperatures. Therefore, a role of 60 in DNA binding is not excluded but is not strongly supported by the present data, in keeping with the lack of detectable 60-DNA cross-linking (28). Schematic models of TFIIIC-DNA complexes based on site-specific cross-linking studies tentatively visualized the 60-kDa polypeptide as being unbound to DNA, connecting the 95 and 138 subunits, and overlapping the A block-B block interval (10). This proposal fits remarkably well with our present observations, since 60 appears to reside at least partly in B and possibly also contact 95, as suggested by the in vivo suppression of 60 mutant lethality by overexpression of 95, although two-hybrid experiments did not confirm this interaction (data not shown).

In view of the position of 60 in the TFIIIC-DNA complex, the effect of a 60 mutation at the level of TFIIIB recruitment was rather unexpected. Since 131 was the only subunit of TFIIIC extending upstream of the transcriptional start site (7), it has been assumed to be entirely responsible for TFIIIB assembly. Indeed, 131 and no other TFIIIC subunit was found to interact with TFIIIB70/Brf1 and TFIIIB90/B" (14, 33, 51). Several lines of evidence, however, indicate that 60 most likely also participates in TFIIIB recruitment: (i) the lethality of the Ntag-60 variant at a nonpermissive temperature was suppressed by overexpression of two TFIIIB components, TFIIIB90/B" and TBP (not by TFIIIB70 though); (ii) the in vitro transcription defect of the mutant TFIIIC was strongly alleviated by increasing TFIIIB concentration and more specifically by increasing TBP concentration (again not TFIIIB70 or B"); and (iii) 60 was found to interact directly with TBP. The possibility of an indirect role of 60 in TFIIIB recruitment could be envisioned in view of its possible interaction with 95 that belongs to the A domain. The 60 mutation could perturb 95-A block binding or affect indirectly, via 95, TFIIIB assembly by 131. If this were the case, one would expect a suppression of the in vivo or in vitro defects by increased TFIIIB70 dosage. Indeed, mutations in TFIIIC (37), in TBP (12, 16), or in promoter elements decreasing factor-DNA interaction (39) were always found to be best suppressed in vivo by overexpression of TFIIIB70. This makes sense since TFIIIB70 interacts with TFIIIC (14, 33), initiates TFIIIB assembly (31), and appears to be limiting for transcription both in whole-cell extracts and in vivo (39, 54, 55). Since increasing the TFIIIB70 concentration did not correct the TFIIIC mutant phenotype in vivo and in vitro, the rate-limiting step caused by the 60 mutation was expected to follow TFIIIB70 recruitment step, i.e., the TBP recruitment step (31). Since it appears unlikely that the 60 mutation would have an indirect effect on the recruitment of TBP by TFIIIB70, we rather favor the idea that 60 is directly involved in TFIIIB recruitment by contacting TBP. The coimmunoprecipitation of TBP and 60 comes in support of this proposal. The interaction of TBP with TFIIIC subunits has not been previously reported. However, Huet et al. (26) have noted that TBP slightly retarded the migration of TFIIIC-DNA complexes on a TATA-less gene. Since TFIIIC specifies the orientation of TFIIIB on the DNA (and thereby the direction of transcription) (60), the direct interaction of TFIIIC with TBP, via 60, might well contribute to fix its correct orientation on the DNA. Alternatively, 60-TBP interaction may not be involved in positioning TBP on the DNA but only represent a transient step facilitating the delivery of TBP to the TFIIIB70-TFIIIC-DNA complex.