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

Figure 4

Three lethal Med6p mutants also show transcriptional defects in a reconstituted in vitro system. Either wild-type (WT) or med6 ts-2 mutant (Ts) Pol II holoenzyme (700 ng each) was preincubated for 10 min at 25°C or 37°C with other supplements containing two DNA templates, GTFs, 0.5 mM ATP, and Gal4-VP16 (30 ng), with or without the indicated recombinant Med6p (80 ng each). After PIC formation, the transcription reaction (30 min, 25°C) was initiated by the addition of [α-³²P]-UTP and CTP. The mRNA products were purified and resolved on 7% denaturing polyacrylamide gel, followed by autoradiogram. Transcription (%) was set to 100% as to the fold activation (signal from GAL4 template divided by signal from GCN4 template) shown by wild-type holoenzyme upon PIC formation at 37°C. The data quantitation was performed with the use of Personal Molecular Imager FX system and associated program (Bio-Rad).