Fig. 3.

Thr4 is required for association of elongation and termination factors. (A) Flowchart of the method to identify factors whose binding is compromised in T4A. (B) Volcano plot illustrating the fold change ln(T4A/WT) and the significance –Log₁₀[false discovery rate (FDR)] of differential binding. Proteins depleted at least one natural log in T4A with an FDR of <0.05 are color coded and labeled as in C. (C) Physical interconnectivity (46) between the significantly depleted proteins identified in B. Gray background indicates low density connectivity. (D) Flowchart of the experimental design to assay binding of factors to human CTD (Top). Phosphorylation state of HeLa extract, unphosphorylated human Rpb1, or Rpb1 phosphorylated with human PLK3 (Left). Enriched hypophosphorylated Pol II (Thr4-CTD) and PLK3-treated CTD (pThr4-CTD) was incubated with the indicated TAP-tagged proteins, expressed in yeast. Indicated proteins were assayed for binding to pThr4-bearing Rpb1 and probed with α-TAP (Right).