Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex

Figure 5

The deficiency of Med6p-Δ2 in WCEs and purified Mediator fraction. (lanes 1–12) WCEs were prepared from YCL4 yeast strains expressing the indicated LexA-Med6p derivatives (pCL-MED6) along with (+) or without (−) coexpression of the native forms of Med6p derivatives (pC-MED6) by copper induction for 2 h. Samples were resolved on an 8% denaturing polyacrylamide gel, and expression levels of LexA-fused and native forms of Med6p derivatives were examined by western analysis using anti-LexA antibody and anti-Med6 antibody, respectively. Western analysis for TBP was done for loading control. The degradation product of LexA-Med6p-Δ2 is indicated by an asterisk. (lanes 13–16) WCEs were prepared from the indicated yeast strains and subjected to DEAE column fractionation to enrich the Pol II holoenzyme. Pol II holoenzyme in each sample was immunopurified with beads-coupled anti-Med14 antibody and subjected to western analysis to measure the amounts of LexA-Med6p derivatives (α-LexA) contained in the immunopurified Mediator fraction (α-Med14).