Figure 6.

(A) Purified pol II from HeLa cells was assayed for binding to GST-E6-AP and Rsp5 (lanes 2 and 3). The largest and second largest subunits were detected by immunoblotting. The input amount of protein was loaded in lane 1. The phosphorylated and underphosphorylated forms of the largest subunit are indicated. (B) In vitro ubiquitination of human pol II large subunit. Total HeLa cell extract was incubated without (−, lane 1) or with a DEAE fraction from insect cells infected with nonrecombinant baculovirus (wtv, lane 2), Rsp5-expressing virus (Rsp5, lane 3), or Rsp5 C-A-expressing virus, along with ubiquitin, ATP, and E1 and UBC8 E2 proteins. The reactions were analyzed SDS/PAGE and immunoblotting with the anti-CTD antibody.