Figure 2.

Ubiquitination of Pol II LS is reduced by template DNA. (Upper) Ubiquitination reactions were done under in vitro transcription conditions and analyzed by SDS/10% polyacrylamide gel electrophoresis followed by Western blotting. XbaI-cut (2 μg) pHIV+TAR-G400 (24) was added as a template DNA. Reaction mixtures were preincubated with 5 μM α-amanitin for 3 min at 30°C followed by incubation with template and 1 mM ATP as indicated. Pol II LS was probed with anti-Pol II LS antibody N20 (α-Pol II LS N20). (Lower) Ubiquitinated Pol II LS was quantified and normalized to lane 2 as 100.