Figure 2

Composition and activity of purified human mediator (hMed) and mediator-depleted nuclear extract (ΔNE). (A) HeLa nuclear extracts were fractionated on a GST-TR_LBD affinity matrix. The starting (lane 1) and depleted extract (lane 2) along with increasing amounts of mediator fraction (lanes 3–5) were fractionated on 4%–15% SDS polyacrylamide gels and analyzed by immunoblot with antibodies against representative subunits of hMed and GTFs. Autoradiographs of the blots are shown. Note that hMed is depleted from the ΔNE. Med220 is depleted greater than 30-fold. Also, general transcription factors, including TBP and TAFs of TFIID, are present in the ΔNE fraction but are not present in the purified hMed fraction. (B) Purified hMed complements ΔNE for activated transcription. Twenty nanograms of pG₅E4T template (lanes 1–6) was incubated with 35 μg ΔNE (lanes 1–4) and (lanes 3,4) or 1 unit purified hMed (see Materials and Methods) (lanes 5,6) in the absence (lanes 1,3,5) or presence (lanes 2,4,6) of 5 ng GAL4-VP16. Transcription was measured by primer extension (arrow). Note that hMed alone cannot support transcription. GAL4-VP16-induced transcription is detected only when purified hMed is supplemented with ΔNE (lane 4). (C) The VP eluate complements ΔNE. pG₅E4T (lanes 1–8) was incubated with 35 μg HeLa NE (lanes 1,2), 35 μg ΔNE (lanes 3–6) and/or 2μg VP eluate (lanes 4–8) in the presence (+) or absence (−) of GAL4-VP16. Note that neither VP eluate nor ΔNE alone support transcription but the combination is active.