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

Figure 6

Artificial recruitment assay and chromatin immunoprecipitation analysis of Med6 mutants. (a) Reporter gene activation by targeted recruitment of Mediator via LexA-fused Med6p derivatives. Yeast strain L40 harboring pCL313-Med6 derivatives were cultured in synthetic glucose media containing 0.5 mM copper. The β-galactosidase activity of chromosomal 8xlexA_op-LacZ reporter gene in cultured cells was measured in triplicate. (b) Chromatin immunoprecipitation for recruitment of Mediator (α-Med14), LexA-Med6ps (α-LexA), and TBP (α-TBP) to promoter regions of chromosomal reporter (8xlexA_op) and GAL1-10 (GAL1-10p) genes. L40 strains expressing indicated pCL313-Med6 derivatives were grown in synthetic media containing glucose (Glc) or galactose plus raffinose (Gal + Raf) for 6 h in the presence of copper. Chromatin fragments were prepared from WCEs and immunoprecipitated with the use of the indicated antibodies. DNAs were purified from immunoprecipitates and subjected to PCR (25 cycles) with the use of oligomers specific to promoter regions of reporter gene (PCR product, 200 bp) or GAL1-10 gene (PCR product, 370 bp), respectively. The amount of DNA used for INPUT PCR corresponds to 1% of the immunoprecipitated DNA samples used in PCR.

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