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. 2001 Feb;21(4):1404–1415. doi: 10.1128/MCB.21.4.1404-1415.2001

FIG. 3.

FIG. 3

The binding of TBP to the pS2 TATA sequence is severely restricted by the in vivo chromatin structure of the promoter. (A) Purified viral DNA containing the pS2 promoter (100 ng, 1.0 nM) was incubated with increasing concentrations of human TBP, at the concentrations (in micromolars) indicated above the individual lanes. Subsequently, the reaction mixtures were digested with DNase I, and the DNA products were amplified by LMPCR (48). TBP preparations were assayed for the percentages of TBP molecules that were active for specific DNA binding as described previously (25). The preparation used for the experiments illustrated in Fig. 3D, 4A, and 5A contained essentially all active protein, while the preparation used for the experiments shown in Fig. 3A to C was approximately 30% as active. A control DNA sample was incubated in the absence of TBP (lanes 1 and 8). Dimethyl sulfate/piperidine-treated SVSpS2 viral DNA provided a G ladder (lane G); the nucleotide positions of the pS2 promoter are indicated to the left of this lane. The position of the TATA box is illustrated by the pS2 sequence TATAAAA. The binding of TBP is evidenced by a region of protection from cleavage as delineated by the bracket to the right. (B) Purified viral DNA containing the pS2 promoter was incubated with TBP at the protein concentrations indicated above the individual lanes but supplemented either with the basal transcription factor TFIIA (+) or with TFIIB (lanes 5 and 6). TFIIA and TFIIB concentrations equaled or exceeded that of TBP; TFIIA concentrations were as follows: lanes 1 and 2, 0.13 μM; lane 3, 0.25 μM; lane 4, 0.63 μM. TFIIB concentrations were as follows: lane 5, 0.14 μM; lane 6, 0.35 μM. The designations are as given for panel A. (C) MCs (230 pM) containing the pS2 promoter were isolated from CMT cells and were incubated with TBP at the concentrations noted. The position of NUC T is diagrammed on the left. The remaining designations are as noted above. (D) MCs were incubated with TBP as described for panel C, with the addition of the basal transcription factor TFIIA. TFIIA concentrations were as follows: lanes 1 to 4, 0.25 μM; lane 5, 0.50 μM; lane 6, 1.0 μM.