Solutions, DNAs, and proteins.
H buffer contained 20 mM HEPES (pH 7.5), 10% glycerol, 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, and the molar KCl concentration indicated in the buffer name. TSB contained 20 mM Tris acetate (pH 8.0), 20% glycerol, 2 mM MgCl2, 200 mM potassium glutamate, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride. Stop mixture contained 3 M ammonium acetate and 125-μg/ml tRNA. PSB contained 125 mM Tris-HCl (pH 6.8), 10% glycerol, 3.1% sodium dodecyl sulfate (SDS), 0.71 M β-mercaptoethanol, and 0.5-mg/ml bromphenol blue.
TI and pG6
TI were previously described (6
). Promoter fragments for in vitro transcription were generated from pG6
), pBRVA1 (45
), and pGEM-U6 (22
) by PCR. The TI promoter fragment was generated by cleavage of pSP72(TATA/Inr) with restriction endonuclease Eco
HeLa nuclear extracts and phosphocellulose fractions were prepared as previously described (30
). The following recombinant proteins were produce in Escherichia coli
and purified as previously described: GST-TBP-N and GST-TBP-C (15
), TBP (31
), yeast Mot1 (4
), and TFIIA (44
). The B-TFIID fraction was obtained from and purified by R. Meyers and P. Sharp (Massachusetts Institute of Technology).
Purification of TAF-172 for internal peptide sequencing.
TAF-172 was immunopurified from 725 mg of the HeLa cell-derived phosphocellulose 0.1 to 0.3 step fraction (gift of D. Reinberg [University of Medicine and Dentistry of New Jersey]) with 2 mg of affinity-purified TBP antibodies as previously described (37
), with the following modifications. TBP immunoprecipitates were washed with H buffer containing 0.1 M guanidine hydrochloride (GuHCl), and TAF-172 was eluted with H buffer containing 1 M GuHCl. The TAF-172 pool (~2 μg) was dialyzed against Tris-EDTA buffer, electrophoresed on an SDS–6% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, stained with amido black, and subjected to tryptic digestion in accordance with standard protocols. Internal peptide sequencing of reverse-phase-purified peptides was performed by the Wistar Protein Microsequencing Facility (Philadelphia, Pa.).
Cloning of TAF-172.
Converging degenerate primers (GARTAYATHGCNGGNGC and GCNGGRTCYTCCATDAT), which encode the terminal regions of the sequenced peptide EVLQEYIAGADTIMEDPATR, were used in a PCR with oligo(dT)-primed human cDNA. PCR products were separated by polyacrylamide gel electrophoresis, and the expected fragment was excised and sequenced. The sequence was used to synthesize the nondegenerate probe GAGTATATTGCGGGTGCCGACACCATCATGGAAGACCCAGC. The probe was 32P end labeled and immediately used to screen a phage λgt10 human cDNA library. An initial partial clone was isolated, and its insert was subcloned into EcoRI-cut pGEM-7z (Promega) and sequenced. The clone was used in subsequent screens to isolate additional clones, one of which contained an open reading frame coding for amino acids 1 to 876 in TAF-172. Sequences in the clone and sequences coding for peptide sequences predicted to be located near the C-terminal end of TAF-172, based on alignments with MOT1, were used in a nested PCR (external primers, AGCCACATCATCTTTCG and CCARTTNACNCCRTCYTG; internal primers, TCGAGTAAACAACAATG and ACNCCRTCYTGYTGRTA) on randomly primed cDNA to obtain a 1.4-kb probe for the 3′ half of the gene. This probe contained an open reading frame coding for amino acids 816 to 1272 in TAF-172. Screens of phage libraries with this probe yielded a partial clone which contained an open reading frame coding for amino acids 976 to 1848 in TAF-172.
TAF-172 sequences encoding amino acids 1 to 623 and 952 to 1858 were subcloned separately into the NdeI site of the pET16b expression vector (Novagen) by PCR. The polyhistidine-tagged proteins were expressed in E. coli BL21 (Novagen) and purified from GuHCl-solubilized inclusion bodies by nickel affinity chromatography (Pharmacia) in accordance with the manufacturer’s directions. Both proteins were injected into the same rabbit to produce polyclonal antibodies. Both proteins were coupled to Affi-Gel 10 (Bio-Rad) and used to affinity purify TAF-172 antibodies. Antibodies were eluted with a solution of 50 mM glycine (pH 2.0) and 150 mM NaCl. The antibodies were immediately neutralized with Tris-Cl (pH 8) and dialyzed against TSB (lacking dithiothreitol). Affinity-purified human TBP and TAFII250 antibodies were purified in the same manner against their cognate antigens.
Baculovirus expression and purification of TAF-172.
To generate full-length TAF-172, the two cDNA clones and the overlapping PCR product were combined by using convenient restriction sites and inserted into the vector pFastBac1 (Gibco-BRL) along with the 6× polyhistidine tag acquired from pET16b. Recombinant baculovirus was generated by using the Bac-to-Bac Baculovirus Expression kit (Gibco-BRL). Sf9 cells (5 × 108) grown in Grace’s insect medium (Gibco-BRL) supplemented with 10% fetal bovine serum were infected with recombinant TAF-172 baculovirus for 72 h and harvested by centrifugation. Cells were washed in 25 ml of PBSM (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 · 7H2O, 1.4 mM KH2PO4, 12.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride) and lysed with 10 ml of AS0.6 buffer (60 mM Tris acetate [pH 8.0], 15 mM MgCl2, 20% glycerol, 0.625 mM ammonium sulfate, 1 mM phenylmethylsulfonyl fluoride, 2-μg/ml leupeptin, 2-μg/μl pepstatin A). The cell lysate was sonicated to reduce viscosity and centrifuged to pellet cell debris. Polyhistidine-tagged TAF-172 was affinity purified on a 2-ml nickel Sepharose column (Pharmacia) in accordance with the manufacturer’s directions and eluted with NE buffer (20 mM Tris acetate [pH 8.0], 10% glycerol, 2 mM MgCl2, 200 mM potassium glutamate, 400 mM imidazole). The eluate (4 ml) was chromatographed on a 150-ml Sephacryl S300 gel filtration column (Pharmacia) equilibrated with TSB. TAF-172 fractions eluting between 180 and 230 kDa (9 ml) were pooled and applied to a 0.5-ml S-Sepharose (Pharmacia) column equilibrated with TSB. TAF-172 was present in the flowthrough fraction and was stored at −80°C.
Rabbit serum (1 ml) containing either TAF-172, TFIIA, or control nonspecific antibodies was incubated with protein A Sepharose (200 μl) for 2 h at 4°C with constant mixing. The resin was washed with 200 mM sodium borate solution, and antibodies were cross-linked to the resin with 10 mM dimethyl pimelimidate at 23°C for 1 h. Cross-linking was quenched with 200 mM ethanolamine (pH 7.5), and the resin was washed with H.1 buffer.
HeLa nuclear extracts (22 μg, 1 ml) were mixed with protein A Sepharose containing cross-linked TAF-172 (0.2 ml) and/or TFIIA (0.5 ml) or equivalent amounts of control antibodies at 4°C for 3.5 h. The resin was removed by centrifugation, and the extracts were stored at −80°C. The resin was washed with H1 buffer, followed by H.1 buffer, and then drained. Depletion of TBP from nuclear extracts was done similarly, except that 0.2 mg of affinity-purified TBP antibodies was used per mg of nuclear extract.
TAF-172 was incubated with either GST-TBP-N, GST-TBP-C, or TSB buffer alone for 2 h at 37°C. Proteins were then incubated with glutathione agarose at 4°C for an additional 1 h and washed with H1 buffer. Bound proteins were eluted with H.15 buffer containing 7.5 mM reduced glutathione. Eluted fractions were precipitated with trichloroacetic acid.
The 50-bp probe contains the adenovirus major late TATA box (TATAAAAG) and 28 bp of DNA upstream of the TATA box (2
). The DNA was 32
P end labeled with polynucleotide kinase and gel purified in accordance with standard protocols. In addition to TBP, TAF-172, and ATP in the amounts indicated in the corresponding figure legend (see Fig. ), reaction mixtures contained 4 mM Tris-Cl (pH 8.0), 4% glycerol, 5 mM MgCl2
, 60 mM KCl, 0.1% Brij 58, 5-μg/ml poly(dG-dC), and 100-μg/ml bovine serum albumin (2
). Reaction mixtures containing TAF-172 or Mot1 and/or human TBP were incubated for 20 min at 23°C prior to loading on the gel. Samples (20 μl) were loaded onto native 6% (59:1 acrylamide-bisacrylamide ratio) polyacrylamide gels containing 1× TG buffer (25 mM Tris-Cl [pH 8.3], 190 mM glycine, 1 mM EDTA, 5 mM magnesium acetate), 2.5% (vol/vol) glycerol, and 0.5 mM dithiothreitol in running buffer containing 1× TG. Electrophoresis was continued at 35 mA for 60 to 90 min at 4°C.
FIG. 5 ATP-mediated dissociation of TAF-172–TBP–DNA complexes as detected by EMSA. (A) Reaction mixtures contained human TBP (hTBP) (lanes 1, 2, and 5 to 15), radiolabeled TATA DNA (50 bp), and increasing concentrations of TAF-172 (lanes 5 to (more ...) ATPase assay.
Reaction mixtures contained TBP, TAF-172, G6TI DNA (361 bp), and ATP (including 0.5-μCi/μl [α-32P]ATP) at the concentrations indicated in the corresponding figure (see Fig. ). Reactions were performed in TSB at 30°C in a volume of 10 μl. At various times, 1-μl samples were spotted on polyethylenimine thin-layer chromatography plates, dried, and developed with a solution of 0.8 M glacial acetic acid and 0.8 M LiCl2. The plates were dried, and the radioactivity present as [α-32P]ATP and [α-32P]ADP was quantitated by a PhosphorImager and NIH Image software. The percent ATP hydrolyzed was plotted as a function of time by using Kaleidagraph software, and a global linear fit of the data was made. Standard errors are reported.
FIG. 6 TAF-172 is a TBP-stimulated DNA-dependent ATPase. (A and B) TAF-172 was assayed for ATPase activity in the presence or absence of TBP and a 361-bp G6TI promoter DNA fragment, as indicated. Background rates of ATP hydrolysis determined in parallel reactions (more ...) In vitro pol II and pol III transcription assays.
In vitro pol II transcription reaction mixtures contained 3 mM HEPES, 9 mM Tris acetate (pH ~7.9), 5 mM MgCl2, 10% glycerol, 15 mM KCl, 90 mM potassium glutamate, 50 μM EDTA, 0.5 mM dithiothreitol, 4 mM spermidine, 1% polyvinyl alcohol, 10-μg/ml poly(dG-dC), 1-μg/ml G6TI 361-bp promoter DNA or 10-μg/ml TI 2,487-bp DNA, 0.5 mM GTP, 0.5 mM UTP, 0.5 mM CTP, 10 μM ATP, and 4 μCi of [α-32P]ATP in a volume of 20 μl. Nuclear extracts (60 μg) and other reaction components (except nucleoside triphosphates) were added, and incubations at 30°C were continued for 20 min. Transcription was initiated by addition of nucleoside triphosphates and allowed to proceed at 30°C for 20 min. Reactions were terminated with 80 μl of stop mixture. The RNA was extracted with a phenol-chloroform mixture, precipitated with ethanol, resuspended in 90% formamide, and electrophoresed on 7 M urea–6% polyacrylamide gels. Gels were dried, and the radioactivity was visualized by using a PhosphorImager. In vitro VA1 pol III transcription reaction mixtures contained 6 mM HEPES, 4 mM Tris acetate (pH ~7.7), 4 mM MgCl2, 7% glycerol, 50 mM KCl, 40 mM potassium glutamate, 25 μM EDTA, 0.5 mM dithiothreitol, 0.5 mM spermidine, 10-μg/ml poly(dG-dC), 1-μg/ml VA1 315-bp promoter DNA, 0.5 mM GTP, 0.5 mM UTP, 0.5 mM CTP, 10 μM ATP, and 4 μCi of [α-32P]ATP. In vitro U6 transcription reaction mixtures contained 6 mM HEPES, 4 mM Tris acetate (pH ~7.7), 4 mM MgCl2, 7% glycerol, 45 mM KCl, 60 mM potassium glutamate, 25 μM EDTA, 0.5 mM dithiothreitol, 1 mM spermidine, 1% polyvinyl alcohol, 10-μg/ml poly(dG/dC), 1-μg/ml U6 306-bp promoter DNA, 0.5 mM GTP, 0.5 mM UTP, 0.5 mM CTP, 10 μM ATP, and 4 μCi of [α-32P]ATP.