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. 2019 Jun 1;33(11-12):705–717. doi: 10.1101/gad.322453.118

Figure 4.

Figure 4.

Ccr4–Not does not directly ubiquitylate RNAPII. (A) Coomassie blue stain of purified Ccr4–Not complex. (B) Not4 autoubiquitylation assay using the Ccr4–Not complex. The Ccr4–Not complex (1.3 µg) was incubated with 50 ng of GST-Ube1 (E1), 50 ng of UbcH5c (E2), and 8 µg of ubiquitin. Reactions were terminated after the times indicated, and Not4 was detected by Western blotting. The minus E2 and E3 samples were incubated for 30 min. (C) Ubiquitylation assay using RNAPII as a substrate. Three-hundred nanograms of RNAPII, 0.6 µg of Ccr4–Not, 50 ng of GST-Ube1, 50 ng of 6xHis-Ubc4, and 8 µg of ubiquitin were used in the assay. Rpb1 was detected by Western blotting using 8WG16. (D) Schematic of the procedure producing RNAPII ECs. Details are described in the Materials and Methods. (E) Ubiquitylation reactions comparing free polymerase and ECs. (Top panel) Free RNAPII and that incorporated into immobilized ECs were incubated with E1 (50 ng of GST-Ube1), E2 (50 ng of UbcH5c), and either 1.8 µg of Ccr4–Not (E3) or, as a control, 75 ng of recombinant GST-Rsp5 (E3). Two of the reactions contained either full-length Def1 or the truncated active form (amino acids 1–500). Rpb1 was detected by Western blotting using 8WG16.