Figure 4.

Mediator depletion inhibits nmt1 Inr transcription. (A) Mediator was depleted from a WCE from a TAP-tagged Med7 strain using IgG-Sepharose beads, and transcription was recovered by replacing different combinations of factors. The WCE from the same TAP-tagged strain was depleted of TAFs using anti-TAF1 antibodies crosslinked to Sepharose beads. It is observed that Mediator depletion completely abolishes transcription of the nmt1 Inr (WCED), and is recovered by replacing an eluate from the IgG-Sepharose beads (WCED + IgG Seph. Elution), with a crude fraction from a P11 chromatographic fraction of WCE (WCED + 0.5 M P11), or with the same fraction plus the elution of the IgG-Sepharose column (WCED + IgG Seph + 0.5 M P11). TFIIB, TBP, RNAPII, or TFIIF cannot complement for the activity eluted from the IgG-Sepharose beads. Importantly, TAF-depletion has no effect on nmt1 Inr transcription (WCE TAP + αTAF1) and an eluate from the anti-TAF1 beads cannot complement Mediator depletion (WCED + TAF1). (B) Western blot analysis with different antibodies was used to test the extension of the depletion of the WCED. It is observed that Mediator was completely depleted (Med17 panel) by IgG Sepharose beads, and TAFs are depleted by anti-TAF1 Sepharose beads (TAF5 panel). Depletion of Mediator does not affect the levels of TAFs, and depletion of TAFs does not affect Mediator levels. Levels of RNAPII and other transcription factors are not affected by either Mediator- or TAF-depletion.