Supplementary Figure 3: C-TIR stabilizes Pol II binding region I interaction with Pol II.

a, Schematic of full-length Gdown1, N-terminal (1–180), C-terminal (181–368), and C-terminal deletion mutants and summary of Pol II binding and in vitro transcription assays. b, Binding of Gdown1 fragments to Pol II. Immobilized GST or GST-Gdown1 proteins were incubated with HeLa nuclear extract in buffer C containing 0.1 or 0.3 M KCl and 0.1% NP-40. After washing with buffer C containing 0.1 or 0.3 M KCl and 0.1% NP-40, bound proteins were analyzed by immunoblot. c–e, In vitro transcription assays. Reactions contained 10 ng of TBP, 10 ng of TFIIB, 25 ng of TFIIF, 10 ng of TFIIEα, 5 ng of TFIIEβ, 50 ng of Pol II or Pol II(G), and the indicated amounts of His-tagged full-length Gdown1 or C-terminal deletion fragments. Reactions were incubated at 30 °C for 1 h, and purified RNA products were resolved by PAGE. f, Pol II binding assay. Full-length Gdown1 and C-terminal deletion fragments were incubated with Pol II in buffer C containing 0.1 M KCl and 0.1% NP-40 for 1 h and subjected to immunoprecipitation with anti-CTD antibody (8W16). Bound proteins were analyzed by immunoblot. g, h, Pol II binding assay. Immobilized GST and GST-Gdown1 proteins were incubated with HeLa nuclear extract in buffer C containing 0.1 or 0.3 M KCl and 0.1% NP-40. After washing, bound proteins were analyzed by immunoblot. i, Pol II binding assay. Immobilized GST and GST-Gdown1 proteins were incubated with purified Pol II in buffer C containing 0.1 M KCl and 0.1% NP-40. After washing, bound proteins were analyzed by immunoblot. j, Schematic of the GST-fused Gdown1 fragments that were used in g–i. Amino acid residues (L303/4) that were changed to alanine by site-directed mutagenesis are shown in a white triangle.