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. 2000 Mar 1;28(5):1199–1205. doi: 10.1093/nar/28.5.1199

Figure 2.

Figure 2

Reconstitution of column fractions derived from mouse whole-cell extract allows specific complex formation with the mouse rDNA core promoter. (A) Protein fractions containing TFIA, Pol I, UBF or SL1, were prepared from mouse whole-cell extract as described in Materials and Methods. The fractions, denoted as ‘+’ above each lane, were incubated with radiolabeled mouse rDNA core promoter fragment, and analyzed in an agarose gel. The amounts of protein added are as follows: TFIA-fraction, 14.8 µg; Pol I-fraction, 2.45 µg; UBF-fraction, 0.86 µg; SL1-fraction, 0.92 µg. The arrowhead indicates the specific complex reconstituted by the TFIA-, Pol I- and SL1-fractions. The lower panel is an autoradiogram of the UV cross-linking assay with the same combination of the fractions used in the agarose gel shift. Positions of a molecular weight marker are indicated on the right. The arrowhead indicates the position of p70. (B) TBP is involved in the reconstituted complex. An agarose gel shift assay was performed with the mouse core promoter probe and the mouse TFIA-, Pol I- and SL1-fraction. Following incubation for the DNA–protein complex formation, 1 µl of affinity-purified anti-TBP antibody (0.68 mg/ml) was added to the reaction mixture (lane 2). The mixtures were further incubated for 30 min at 30°C and then subjected to agarose gel electrophoresis. The arrowhead indicates the specific complex containing TBP.