Figure 3.

(A) In vitro ubiquitination of Rpb1. In vitro-translated Rpb1 was incubated without (−, lane 1) or with a DEAE high-salt fraction derived from insect cells infected with nonrecombinant baculovirus (wtv, lane 2), Rsp5 virus (lane 3), or the C-A mutant (lane 4), along with recombinant E1 and E2 (UBC8) proteins, ATP, and ubiquitin. The migration position of full-length Rpb1 is indicated, as is multi-ubiquitinated Rpb1 [Rpb1-ub_(n)]. (B) Ubiquitination of GST-CTD. In vitro-translated GST (lanes 1–3) or GST-CTD (lanes 4–6) were incubated with a control protein fraction (lanes 1 and 4), Rsp5 (lanes 2 and 5), or Rsp5 C-A (lanes 3 and 6), as in A. The ubiquitinated GST-CTD species are indicated.