Analysis of the putative HMG domains of TAF130 homologues. Sequences corresponding to the HMG regions of several TAFs were aligned by using the program CLUSTAL W (EMBL Outstation) and compared to the original alignment between human TAF250 and the HMG-1.B (44) and to the HMG consensus (13). The three α-helices that form the HMG domain are depicted above the TAF sequences. Residues shaded in gray are conserved among the TAFs, and boxed residues are similar between human TAF250 and HMG-1.B.

Interaction of TFIID with activation domains. Whole cell extracts containing (HA)₃-tagged wild-type TAF130 or TAF130-Δ16 were incubated with glutathione-Sepharose beads bound to GST protein (G) or to GST fusions to a mutated form of the VP16 activation domain (Δ), the GST-VP16 activation domain (V), or the TADIV activation domain of Adr1 (T). The indicated TFIID components in the bound (left panel) or supernatant (right panel) fractions were detected by Western blotting with the indicated antibodies.

The TAF130-Δ16 derivative is transcriptionally inactive. (A) RNA levels of the indicated genes in strains containing a copper-inducible TAF130-depletion allele and plasmids expressing (HA)₃-tagged wild-type TAF130 or TAF130-Δ16 at 0, 2, and 4 h after the addition of copper. The TRP3, RPL9A, and RPS8A genes are dependent on TAF130 function, whereas the PGK and tRNAw genes are not (32, 45). (B) Levels of wild-type and mutant TAF130 proteins at the indicated times after addition of copper, as determined by Western blotting with antibodies to the HA epitope or to TAF130. The position of the ubiquitin-tagged wild-type TAF130 (UbWT), which is degraded upon copper addition is also indicated.

Promoter association of TAF130 derivatives in vivo. (A) Expression levels of the indicated (HA)₃-tagged TAF130 derivatives expressed from the natural TAF130 promoter on centromeric plasmids as assayed by Western blotting using antibodies to the HA epitope or to TAF130. (B) TFIID complex formation by the indicated TAF130 derivatives. (HA)₃-tagged TAF130 derivatives were immunoprecipitated, and components of the TFIID complex were detected with the indicated antibodies. (C) Cross-linked chromatin preparations from strains carrying the indicated (HA)₃-tagged TAF130 derivatives or vector alone (the left panel represents the input sample) were immunoprecipitated with monoclonal antibodies against the HA tag (central panel) or polyclonal antibodies against TBP (right panel). PCR products corresponding to the indicated promoters or the POL1 structural gene are shown.

Promoter binding experiments. (A) Whole cell extracts containing (HA)₃-tagged wild-type TAF130 or TAF130-Δ16 were incubated with paramagnetic bears containing a 500-bp fragment containing a derivative of the HIS4 promoter with one Gal4 binding site (P), the same HIS4 promoter fragment with several mutations at the TATA element (−T), a 280-bp promoterless fragment from the pBluescript vector present in the two previous constructs that contains the Gal4 binding site (L), and the beads without DNA (lane B). The left panel shows the indicated proteins bound to the DNA, whereas the right panel shows the analysis of 10% of the first supernatant after incubation. (B) Effect of the Gal4-VP16 activator on TFIID binding. The promoterless template containing the Gal4 binding site was incubated with extracts containing (HA)₃-tagged wild-type TAF130 or TAF130-Δ16 in the absence (−) or presence (+) of Gal4-VP16.

A dominant-negative TAF130 mutant that forms a normal TFIID complex. (A) Growth of yeast cells containing the plasmid vector or derivatives bearing wild-type TAF130 or the TAF130-N447 mutant expressed from the GAL1 promoter on plates containing glucose or galactose as the sole carbon source. (B) Location of dominant-negative mutations (vertical arrows) with respect to structural and functional features of TAF130. These features include a conserved central domain that has histone acetylase activity (31), a putative acetyl coenzyme A binding site (10), a putative HMG domain (44), an N-terminal inhibitory region (TAND) that strongly interacts with TBP (22, 29), an additional region interacting with TBP (2), and a region interacting with other TAFs (2). (C) TFIID complex formation. HA-tagged wild-type TAF130 or TAF130-N447 was immunoprecipitated from cell extracts with anti-HA antibodies, and components of the TFIID complex were detected by Western blotting with the indicated antibodies. (D) Dominant-negative effects due to overexpression of C-terminal deletions mutants of TAF130 (number indicates last residue of the protein). Growth phenotypes were determined on galactose medium and are defined in arbitrary units with a value of 5 indicating normal growth and a value of 0 indicating no growth. The structures of TAF130-Δ4 (lacks residues 208–303) and TAF130-Δ16 (lacks residues 913 to 1037) are also indicated. The asterisk denotes the N447 mutant isolated in the genetic screen.