Figure 4.

Mediator is required for TFIIH-dependent pol II CTD phosphorylation within the PEC; oncogenic mutations within p53AD (p53QS) prevent activation of stalled pol II. (a) Immobilized template assays with the native HDM2 promoter. Purified PEC factors (TFIIA, IIB, IID, IIE, IIF, IIH, Mediator, and pol II; see ref. 20) were allowed to assemble on the HDM2 promoter; unbound factors were removed by washing, at which time rNTPs were added (including [³²P]-ATP) to visualize and quantitate pol II CTD phosphorylation. Data shown follows a 6-minute incubation with rNTPs; radio-labeled bands migrated around 250 kDa, the approximate size of Rpb1. Bar graph represents mean and s.e.m. from multiple independent experiments (n = 6, 10, 4 for lanes 4–6, respectively). Occupancy of PEC components was measured by western blot, several of which are shown below the bar graph. PECs lacking only one specific component (TFIIH, pol II, or Mediator: lanes 1–3) indicate a Mediator requirement for pol II CTD phosphorylation. (b) and (c) ChIP data reveals that phosphorylated forms of pol II are diminished within the HDM2 gene in the absence of an intact p53AD. (d) and (e) Occupancy of pol II CTD kinases CDK9 (a component of P-TEFb) and CDK8 are not negatively impacted by p53AD mutations. Note that occupancy of another major pol II CTD kinase, TFIIH, is also not altered by p53AD mutations, as observed by ChIP in Figure 1c. The probe location in red (B) represents the promoter/transcription start site. *ChIP output was normalized to WT p53, typically from primer C. For clarity, error bars are not shown but can be viewed in Supplementary Figure 8.