Dimerization of TFIID when not bound to DNA

Science

Volumn 272, Issue 5266, 1996, Pages 1331-1333

  Taggart, Andrew K P ^a   Pugh, B Franklin ^a  

^a PENNSYLVANIA STATE UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

TATA BINDING PROTEIN; TRANSCRIPTION FACTOR;

ARTICLE; BINDING COMPETITION; CONTROLLED STUDY; CROSS LINKING; DIMERIZATION; DNA BINDING; EUKARYOTE; HELA CELL; HUMAN; HUMAN CELL; IMMUNOPRECIPITATION; PRIORITY JOURNAL; PROMOTER REGION;

EID: 0029896209     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5266.1331     Document Type: Article

References (21)

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12. A. K. P. Taggart, A. J. Jackson, B. F. Pugh, date not shown; K. Kato et al., Nucleic Acids Res. 22, 1179 (1994).
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15. 2, 4 mM spermidine, 0.1 mM EDTA, 5% glycerol, 75 mM potassium glutamate, 0.01% nonidet-40, bovine serum albumin (BSA, 5 μg/ml), 9% (v/v) dimethyl sulfoxide, and either 0.1 mg of P.7 and 25 ng of heparin in a volume of 50 μl or 28 ng of recombinant human TBP and 1 μg of polydeoxyguanylic-deoxycytidylic acid [poly(dG-dC)] in a volume of 10 μl. Similar results were obtained when poly(dG-dC), heparin, or BSA were omitted. BMH (Pierce; 0.1 mM for 30 s for TBP, 1 mM for 90 s for P. 7) was included where indicated. TBP reactions were quenched by addition of PSB [375 mM tris-Cl (pH 6.8), 3% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.05% bromphenol blue]. P.7 reactions were quenched with 0.25 M β-mercaptoethanol, and the proteins were precipitated with trichloroacetic acid (TCA) and resuspended in PSB. Samples were subjected to SDS-PAGE on a 7.8% gel, and TBP was visualized by protein immunoblotting (alkaline phosphatase method) with TBP-affinity-purified anti-TBP.
16. Proteolytic fingerprinting was performed on either 1.5 μg of cross-linked recombinant TBP or 3 mg of cross-linked P.7 that had been subjected to SDS-PAGE, transferred to nitrocellulose, and the 90-kD region excised according to F. M. Ausubel et al., Eds., Current Protocols in Molecular Biology (Wiley, New York, 1994).
17. II250 (248C) antigen. Bound proteins were eluted with PSB and subjected to SDS-PAGE on a 7.8% gel. TBP was detected by protein immunoblotting with biotinylated TBP-affinity-purified anti-TBP. Proteins were visualized with streptavidin-horseradish peroxidase (Pierce) and enhanced chemiluminescence (ECL, Amersham).
18. P.7 (0.1 mg) was cross-linked as described (10), except that the heparin was replaced with 2.5 μg of poly(dG-dC) and herring sperm DNA, as indicated. Where indicated, reactions also contained 2 nmol of either TATA (upper strand, 5′ -GGAATTCGGGC-TATAAMGGGGGATCCG-3′) or mutant TATA (upper strand, 5′-GGAATTCGGGCTAAGAAAGGGG-GATCCG-3′) double-stranded oligonucleotides, and were incubated at 30°C for 1 hour before adding BMH.
19. II250.
20. 2, 0.1 mM DTT, and 0.2 mM phenylmethylsulfonyl fluoride] to a final GuHCl concentration of 1 M. Precipitates were removed by centrifugation at 30,000g for 30 min at 4°C1 and supernatants were precleared by passage over columns containing 0.2 ml of nonspecific serum covalently cross-linked to 0.2 ml of protein A-sepharose (Pharmacia) [E. Harlow and D. Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988]. Column flow-throughs were incubated with 0.03 ml of anti-TBP serum covalently cross-linked to 0.03 ml of protein A-sepharose overnight at 4°C with constant mixing. The resin was pelleted by centrifugation and washed twice with ∼300 bed volumes of HO buffer containing 1 M GuHCI and 0.005% nonidet-40, followed by a wash with HO buffer lacking GuHCI. Bound proteins were eluted with PSB and subjected to SDS-PAGE on a 7.8% gel. TBP was detected by protein immunoblotting (ECL method).
21. Supported by grants from NIH (GM47855) and the Searle Scholars Program/Chicago Community Trust. We thank R. A. Coleman for his efforts on the initial phase of the project and for Fig. 2B, and A. Jackson-Fisher for help with tissue culturing. We are especially grateful to R. Tjian for his encouragement.