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. 2000 Mar;74(5):2459–2465. doi: 10.1128/jvi.74.5.2459-2465.2000

FIG. 5.

FIG. 5

FIG. 5

Effect of Rep78 on the complex formation of TBP and the HPV-16 p97 core promoter. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of GST and GST-Rep78 proteins. The full-length AAV rep gene from nucleotide 321 to 2193 was amplified by PCR and cloned into vector pGEX-5X-3 (Pharmacia Biotech, Uppsala, Sweden) for expression of GST-Rep78 fusion protein. The protocol used for expression and purification of GST-Rep78 was as described by the manufacturer (Pharmacia). Lanes 1 and 3, GST-Rep78 and GST proteins eluted with 10 mM glutathione (reduced form); lanes 2 and 4, GST-Rep78 and GST protein conjugated on glutathione-Sepharose 4B beads. (B) EMSA analysis of the influence of GST-Rep78 on the interaction between TBP and the HPV-16 p97 TATA box. EMSA was performed in a total volume of 20 μl of reaction buffer containing 20 mM HEPES-KOH (pH 7.9), 25 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 100 μg of bovine serum albumin per ml, 10% glycerol, and 0.025% Nonidet P-40. Twenty nanograms each of human recombinant TBP (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) and GST-Rep78 at the indicated concentrations was incubated with a 32P-labeled double-stranded 16TATA oligonucleotide (5′-AACGGTTAGTATAAAAGCAGACA), as described by Bauknecht and Shi (3), at 30°C for 30 min. The DNA-protein complexes were resolved on a 5% native polyacrylamide gel in 1× Tris-borate-EDTA (99 mM Tris base, 99 mM boric acid, and 2 mM EDTA [pH 8.3]) running buffer. Lane 1, free 32P-labeled 16TATA oligonucleotide; lanes 2 and 8, TBP-16TATA complexes; lanes 3 to 6, addition of GST-Rep78 to a reaction solution containing preformed TBP-16TATA complexes (assay A); lanes 9 to 11, incubation of TBP with GST-Rep78 prior to addition of 32P-labeled 16TATA oligonucleotide (assay B); lanes 7 and 12, addition of 435 and 145 ng of GST as a negative control for assays A and B, respectively. “G” represents GST and “Probe” represents a free 32P-labeled 16TATA oligonucleotide. The radioactivities of TBP-16TATA complexes on the gel were scored with an instant imager as described for Fig. 1. The percent TBP-16TATA complex formation was calculated as follows: (radioactivity of TBP-16TATA complex in the presence of GST-Rep78/radioactivity of TBP-16TATA complex in the presence of GST) × 100. I, II, and III, TBP-16 TATA complex.