FIG. 2.

Both the N terminus and the C terminus of ICP27 must be intact for binding to RNAP II CTD in vitro. In vitro binding assays were performed with GST-CTD and WT or mutant forms of ICP27 that were translated in vitro. (A) A schematic representation of the ICP27 coding sequence showing the sites of the mutations. The leucine-rich region (LRR), the nuclear localization signal (NLS), the RGG-box motif (RGG), the arginine-rich region 2 (R2), three predicted KH domains (KH1, KH2, KH3), and a zinc finger-like domain (CCHC) are depicted. (B) In vitro binding assays were performed with unphosphorylated (unphos) GST-CTD and WT or mutant forms of ICP27 as indicated. Luciferase was included as a negative control. Input ³⁵S-labeled proteins are shown in the lower panel. (C) GST-CTD was phosphorylated (phos) in vitro with [γ-³²P]ATP by cdc2 kinase, and binding assays were performed with WT and mutant forms of ICP27. Input ³⁵S-labeled proteins are shown in the lower panel. DNase and RNase were added to all binding reactions. Luc, luciferase.