Tau91, an essential subunit of yeast transcription factor IIIC, cooperates with tau138 in DNA binding

Abstract

Transcription factor IIIC (TFIIIC) (or tau) is a large multisubunit and multifunctional factor required for transcription of all class III genes in Saccharomyces cerevisiae. It is responsible for promoter recognition and TFIIIB assembly. We report here the cloning and characterization of TFC6, an essential gene encoding the 91-kDa polypeptide, tau91, present in affinity-purified TFIIIC. Tau91 has a predicted molecular mass of 74 kDa. It harbors a central cluster of His and Cys residues and has basic and acidic amino acid regions, but it shows no specific similarity to known proteins or predicted open reading frames. The TFIIIC subunit status of tau91 was established by the following biochemical and genetic evidence. Antibodies to tau91 bound TFIIIC-DNA complexes in gel shift assays; in vivo, a B block-deficient U6 RNA gene (SNR6) harboring GAL4 binding sites was reactivated by fusing the GAL4 DNA binding domain to tau91; and a point mutation in TFC6 (tau91-E330K) was found to suppress the thermosensitive phenotype of a tfc3-G349E mutant affected in the B block binding subunit (tau138). The suppressor mutation alleviated the DNA binding and transcription defects of mutant TFIIIC in vitro. These results indicated that tau91 cooperates with tau138 for DNA binding. Recombinant tau91 by itself did not interact with a tRNA gene, although it showed a strong affinity for single-stranded DNA.

FIG. 1

Sequence analysis of TFC6 and characterization of anti-τ91 antibodies. (A) Deduced amino acid sequence of TFC6. The four microsequenced peptides used for database searching are underlined (GenBank no. U28372). The TFC6 mutation, E330K, responsible for tfc3-G349E suppression is boxed. (B) Amino acid analysis of τ91. The acidic-basic map shows the position of acidic (A) (D, short bars; E, long bars), and basic (B) (R, long bars; K, short bars; H, smallest bars) amino acids. In the Cys-His map, histidine and cysteine residues are plotted as long and short bars, respectively. (C) Characterization of anti-τ91 antibodies by Western blotting. Lane 1, the polypeptide content of a tDNA affinity-purified TFIIIC fraction was analyzed in a silver-stained SDS–8% polyacrylamide gel. (Lanes 2 and 3, ∼100 ng of TFIIIC (lane 2) and ∼20 ng of purified rTFC6p (rτ91) (lane 3) were subjected to SDS-PAGE, electroblotted to a nitrocellulose membrane, and probed with anti-τ91 antibodies. Immune complexes were visualized with an ECL kit (Amersham). The positions of 91- and 75-kDa proteins are indicated. The amino acid sequence, acidic-basic map, and Cys-His map are printouts from DNA Strider 1.2 software (37).

FIG. 2

Activation of the UAS_G-U6 RNA templates by GAL4(1-147)-τ91 and GAL4(1-147)-τ95. (A) Diagram of UAS_G-U6 RNA gene activation by GAL4(1-147)-τ91. Each template was named according to the position of the upstream activating sequences (UAS_G) in each B block-deficient U6 template. TFIIIC is a chimeric τ factor whose wild-type τ91 subunit has been replaced by the GAL4(1-147)-τ91 fusion protein. Chimeric TFIIIC binds the U6 template through the UAS_G sites, and this allows the factor to bind the A box of the SNR6 gene. TFIIIC then recruits TFIIIB, which directs transcription by Poly III. (B) The UAS_G-U6 RNA transcripts (U6 maxi₂ and U6 maxi₁) and the endogenous U6 RNA from cells transformed with pAS91 (lanes 1 to 4) or pAS95 (lanes 5 to 8) were monitored by Northern blot analysis and autoradiography. The UAS_G binding domain (amino acids 1 to 147) of GAL4 is fused to τ91 in pAS91 or to τ95 in pAS95.

FIG. 3

TFC6 encodes a subunit of factor τ. DNA affinity-purified factor TFIIIC was complexed with the tRNA₃^Glu gene, and, when indicated, the complex was digested with 10 ng of α-chymotrypsin for 10 min at 25°C to generate the fast-migrating complex τB-tDNA. Complexes were then incubated with increasing amounts of polyclonal antibodies directed against τ91 for 45 min at 25°C. Protein-tDNA complexes were analyzed by electrophoresis. τ, position of τ-tDNA complexes; τ-IgG, band retarded upon antibody binding; τB, protease-resistant domain of TFIIIC bound to tDNA. Lanes 1 to 8, no protease; lanes 9 to 12, α-chymotrypsin treatment. Lane 1, no TFIIIC factor, lanes 2 and 9, no antibody, lanes 2 to 7 and 9 to 12, anti-τ91 immunoglobulin G (IgG). Lane 8, control mouse antibodies.

FIG. 4

Temperature sensitivities of mutant, suppressor, and wild-type TFIIIC. Ultrogel-heparin-purified mutant (Mut) (tfc3-G349E) (•), suppressor (Sup) (tfc3-G349E/TFC6-E330K) (▪), and wild-type (WT) (▴) TFIIIC fractions (∼0.7 μg in 2 μl) were preincubated in standard binding or transcription buffer at the indicated temperatures for 10 min before their DNA binding or in vitro transcription activity was tested. (A) DNA binding activities of TFIIIC mutants. TFIIIC fractions were incubated with a tRNA₃^Glu gene probe at 25°C for 15 min, and the complexes were analyzed by gel retardation electrophoresis. (B) Complex formation was quantified in a PhosphorImager and is shown as the percentage of complexes formed after preincubation at 20°C. (C and D) Transcription activities of TFIIIC mutants. The tRNA₃^Leu gene from plasmid pGE2 was transcribed in vitro at 25°C for 45 min in the presence of TFIIIC, recombinant TFIIIB70 and TBP, B", and Pol III. Transcripts were separated in a polyacrylamide gel (C) and quantified in a PhosphorImager (D). In panel D, data are shown as percentages of tRNA₃^Leu synthesis after preincubation of TFIIIC at 25°C.

FIG. 5

Nucleic acid binding activity of rτ91 protein. About 2 ng of highly purified rτ91 protein was incubated with the different nucleic acid probes in 15-μl mixtures containing 20 mM Tris-HCl (pH 8.0), 120 mM KCl, 20 μg of bovine serum albumin, 10% glycerol, 10 to 20 fmol (10,000 cpm) of ³²P-5′-end-labelled nucleic acid probe, and, when indicated, 2.5 mM MgCl₂. After 15 min of incubation at 25°C, rτ91-nucleic acid complexes were separated from free DNA or tRNA by nondenaturing electrophoresis on an 8% polyacrylamide gel. The nucleic acid probes were 20 fmol of native spleen tRNA^Met (tRNA) (lanes 1 to 3), 20 fmol of 200-nucleotide-long single-stranded tRNA₃^Glu gene (ssDNA) (lanes 4 to 6), and 10 fmol of 200-bp double-stranded tRNA₃^Glu gene (ds DNA) (lanes 7 to 9). The arrowhead indicates the rτ91-tRNA^Met complex, and arrows point to the rτ91–single-stranded tDNA complexes.