Fig. 2

Cdk7 strongly stimulates co-transcriptional capping in the purified enzyme system. a 21mers were synthesized in parallel reactions with unlabeled (upper panel) or radiolabeled (lower panel) ribonucleoside triphosphates in the presence of DMSO (−) or increasing amounts of THZ1; reactions were stopped after 15 or 60 min. Upper panel, reaction products were analyzed by western blotting using antibodies against Ser5-phosphorylated Rpb1 (α-pSer5-CTD); two different exposures of the same image are shown. As a control for equal loading of Pol II in each lane, the same blot was probed with antibodies against Rpb2. Lower panel, radiolabeled transcripts were analyzed on denaturing gels and detected by phosphorimaging. b Washed transcription complexes containing 23mers synthesized with or without 150 µM THZ1 were incubated for 4 min with GTP and increasing amounts of capping enzyme (CE). c Graph shows mean and range of two independent reactions performed as in b. d As diagrammed on the left, transcription complexes containing 21mers were prepared in the presence of DMSO or 100–150 µM THZ1, washed, and incubated for 15 min with 50 µM ATP and 100 µM 3′OMeG, with or without 3 ng of capping enzyme. For +TFIIH add-back reactions (lanes 5–8), 300 ng of purified TFIIH was added with capping enzyme. Reactions were assayed for Pol II CTD phosphorylation status by western blotting (top) or for RNA capping (bottom). % Capped indicates average of two independent reactions