Conservation of the structure of seven orthologs of τ131 in four yeasts and three higher eukaryotes. (A) The sequence of S. cerevisiae (TFC4/YGR047c) (39) and the ortholog sequences of K. lactis (this work, GenBank accession number AF229182), S. pombe, and C. albicans (see Materials and Methods), C. elegans (11), D. melanogaster (2), and H. sapiens (21) are schematically presented. TPR motifs were located in the sequences and rated by using sequence information extracted from 200 TPR motifs of S. cerevisiae (Dumay-Odelot and Marck, unpublished). The light- to dark-blue color gradient indicates the fit to the TPR consensus (low to high). The bHLH-Zip motif, previously postulated in the S. cerevisiae sequence (39) is considered fortuitous, as it is not discernible in the six other sequences. Regions outside TPR units are color coded as follows: green, highly variable regions; orange, regions of good conservation between yeasts only; yellow, region of faint conservation in higher eukaryotes only; red, region of high conservation between all seven sequences. (B) Sequences of the second TPR unit in the seven sequences. Amino acids above sequences indicate the 11 best-conserved positions of the TPR motif as derived from 200 S. cerevisiae TPR motifs (Dumay-Odelot and Marck, unpublished). At these positions, conserved residues are indicated by boldface uppercase letters. Conserved residues at other positions are indicated by boldface lowercase letters.

Deletion analysis of τ131. The motifs noted in the τ131 protein (39) have been deleted one by one or in combination as described by Chaussivert et al. (12). Centromeric plasmids harboring various mutant copies of TFC4, expressed from its own wild-type promoter, were tested for their ability to confer viability, at 30 or 37°C, after the shuffling of the URA3 plasmid-borne wild-type copy, in the context of a chromosomal copy of TFC4 partially (65%) disrupted (strain YCK107) (12, 39) or of a totally deleted TFC4 gene (strain YHD3, this work). Phenotypes are indicated as lethal (−), wild type (+), temperature sensitive (ts), or not determined (nd).

Phenotype of TFIIIC mutant factor with TFC4-TPR2 mutant gene borne on centromeric or multicopy plasmids. (A) The ΔTPR2 mutation was constructed as described by Chaussivert et al. (12). A centromeric plasmid harboring the ΔTPR2 deletion mutant copy of TFC4, expressed from its own wild-type promoter, was tested at 30 or 37°C in selective medium for its ability to confer viability in the absence of TFC4 chromosomal copy. The ΔTPR2 mutation, when borne on a centromeric plasmid, confers a thermosensitive phenotype to yeast cells. (B) The phenotype of cells transformed by a multicopy plasmid harboring the ΔTPR2 mutant copy of TFC4 was tested in a wild-type TFC4 context, and when overexpressed, TFC4-ΔTPR2 was found not to have a dominant negative phenotype, even at 37°C. The loss of the wild-type copy of TFC4 on 5-FOA plates was lethal at 30°C, showing that TFC4 was responsible for the cell viability at this temperature (results not shown).

In vitro transcriptional activity of mutant TFIIIC-ΔTPR2. (A) Deletion of TPR2 does not affect the transcriptional start site. The plasmids used as templates (Leu-45, Leu-34, Leu-28, and Leu-21) harbor various versions of tRNA₃^Leu gene with different A block-to-B block distances of 45, 34, 28, and 21 bp, respectively (4). Transcriptions were performed in the presence of Mono Q-purified fractions of wild-type or mutant TFIIIC (100 and 500 ng, respectively), rTBP, rBrf1, partially purified B” fraction, and RNA polymerase III (see Materials and Methods). (B) Mutant TFIIIC is defective for the SUP4 tRNA^Tyr in vitro synthesis. For each experiment, 100 ng of wild-type TFIIIC and 500 ng of mutant TFIIIC Mono Q fraction were preincubated for 10 min at 25 or 40°C (similar amounts of wild-type and mutant TFIIIC were used, based on Western blot experiments). Single-round and multiple transcriptions were performed at 25°C as described in Materials and Methods. The products were separated on a 7 M urea, 6% polyacrylamide gel and revealed by autoradiography.

Compared DNA binding properties of mutant TFIIIC-ΔTPR2 and wild-type factors. (A) Differential salt sensitivity of mutant and wild-type TFIIIC-DNA complex formation. DNA binding reactions containing partially purified wild-type TFIIIC or TFIIIC-ΔTPR2 (in identical amounts) were carried out at 25°C for 10 min in the presence of various concentrations of KCl, ³²P-labeled tRNA₃^Leu gene, and 200 ng of competitor DNA (pBluescript-SK). The concentrations of monovalent cations (K⁺ and NH₄⁺) indicated take into account the ammonium sulfate brought by the protein fractions (25 mM final concentration). Analysis was done by gel retardation assay as described in Materials and Methods. (B) TFIIIC-DNA complex formation after preincubation of TFIIIC at different temperatures. Mutant or wild-type TFIIIC fractions were preincubated for 10 min at 25, 40, or 45°C as indicated in standard transcription buffer and further incubated in 180 mM monovalent cations with ³²P-labeled tRNA₃^Glu gene and pBluescript-SK competitor DNA (200 ng) for 15 min at 25°C.

Heparin resistance of preinitiation complexes containing wild-type or mutant TFIIIC. Wild-type or mutant TFIIIC were preincubated for 10 min at 25 or 40°C as indicated. Biotin-coupled SUP4 tRNA^Tyr gene was then reacted with streptavidin beads (Dynabeads), and preinitiation complexes were formed by addition of rBrf1, rTBP, and rB" onto immobilized DNA. After 60 min at 25°C, heparin (20 or 50 μg/ml) was added and incubation was pursued for 5 min. Beads were then washed to remove TFIIIC, and single-round transcription on SUP4 tDNA^Tyr was performed as described in Materials and Methods. The products were run on a 7 M urea, 6% polyacrylamide gel and revealed by autoradiography. The signal obtained for the mutant factor (left) is weaker than that obtained with the wild-type factor (right), as less protein was used due to a salt concentration limitation.

In vivo suppression of the τ131-ΔTPR2 mutation and other ΔTPR mutations by overexpression of TFIIIB components. Multicopy suppression of τ131-ΔTPR2 by TFIIIB components. Stationary-phase cultures of mutant cells harboring the different multicopy plasmids indicated were diluted 10⁻¹-, 10⁻²-, 10⁻³-, and 10⁻⁴-fold in water and spotted (5 μl) on solid yeast extract-peptone-dextrose medium and grown for three days. The different genes harbored on the high-copy-number vector pFL44L are as follows: pLR30, Brf1; pL1, TBP; pJR38, B”.

In vitro suppression of the τ131-ΔTPR2 mutation by overdosage of B” or Brf1. (A) In vitro suppression assay of the τ131-ΔTPR2 mutation by B”. Transcription mixtures contained identical amounts of wild-type and mutant TFIIIC (Mono Q fraction) preincubated for 10 min at 40°C; plasmid DNA harboring SUP4 tDNA^Tyr, RNA Pol III fraction (50 ng), rTBP (250 ng), rBrf1 (1.2 μg, 30 ng/μl), and various amounts of B” (partially purified B” fraction), as indicated. The final concentration of monovalent cations (K⁺ and NH₄⁺) was kept constant (at 110 mM) by taking into account the ammonium sulfate brought by the protein fractions. Transcripts were analyzed by electrophoresis and autoradiography (left panel). Transcripts were quantified using a PhosphorImager device and ImageQuant software (Molecular Dynamics). Arbitrary units are used to quantify transcription efficiency. Gray bars, wild-type TFIIIC; solid bars, mutant TFIIIC (right panel). (B) In vitro suppression assay of the τ131-ΔTPR2 mutation by Brf1. The same transcription mixtures as for panel A were used, with B” concentration set at 45 ng/μl and various amounts of rBrf1. The two autoradiograms for wild-type and mutant TFIIIC have been obtained from the same gel with two different exposures (the fraction of the gel showing mutant-factor-directed transcription is overexposed). Arbitrary units are used to quantify transcription efficiency. Gray bars, wild-type TFIIIC; solid bars, mutant TFIIIC (right panel).

Physical interaction between τ131 and B”. Interaction between coexpressed τ131 and B” was revealed by copurification assay. High Five cells were coinfected with three different combinations of two recombinant baculoviruses, one expressing a GST-fused subunit (GST-τ131 or GST-Gea1) and the other expressing 8His-B” or calmodulin-TBP. Extracts were prepared as described in Materials and Methods. A glutathione purification was performed, and 50 μl of eluted fractions obtained were loaded onto SDS-PAGE gels. (A) Coomassie blue staining of an 8% polyacrylamide gel. Lanes 1 and 5 are molecular mass markers; some masses are indicated on the left (in kilodaltons). Combinations of recombinant baculoviruses are indicated at the tops of lanes 2, 3, and 4. Lane 6 is a control showing 8His-B” purified to homogeneity (heparine hyper-D and IMAC resin). The positions of B” are indicated with dots in lanes 2 and 6. (B) Input (10 mg of crude extracts) and bound proteins after glutathione purification (50 μl of eluted fractions) were analyzed by Western blotting. The membrane was probed with polyclonal antibodies directed against 8His-B” and TBP as indicated on the left. Lane 7 is the same control as that shown in lane 6 of panel A.