FIGURE 3.

ATPase activity of the 19 S base is required for recruitment of Mediator to the GAL1 UAS. A, Mediator is dispensable for recruitment of the 19 S base to the GAL1 UAS. Both the wild type (WT) and srb4-ts mutant strains expressing Myc-tagged Rpt6p were grown in YPG at 23 °C up to an A₆₀₀ of 0.85 and then transferred to 37 °C for 1 h before cross-linking. Immunoprecipitation was performed as in Figs. 1, B and C. B, ATPase activity of the 19 S base is essential for recruitment of the Mediator complex to the GAL1 UAS. Both the wild type and rpt1-K256R point mutant strains expressing Myc-tagged Srb4p were grown and cross-linked as in Fig. 2A. Immunoprecipitation was performed as in Figs. 1, B and C. C, shown is Western blot analysis. The wild type and rpt1-K256R mutant strains were grown as in panel B. The whole cell extracts from both the wild type and mutant strains were prepared as in the ChIP assay. The whole cell extracts were analyzed by Western blot assay using the anti-Myc and anti-histone H3 antibodies against Myc-tagged Srb4p and histone H3, respectively.