Fig. 8

Release of nascent RNA from ternary complex prevents capping activation. a (Top) Diagram of reaction scheme. Ternary complexes initiated from the AdML promoter were walked to position +222, washed, resuspended in buffer with 50 µM GTP, and treated with T1 RNase for 10 min post capping or pre-capping. When T1 was added after capping, reactions were incubated with 5 ng of capping enzyme for 2 min, then incubated with T1 RNase and 10 mM EDTA for 10 min. When T1 was added before capping, reactions were incubated with T1 RNase for 10 min, then incubated with the indicated amounts of capping enzyme for 2 min. (Bottom) Diagram of the RNA sequence associated with ternary complexes, showing T1 cleavage sites (scissors). Purple indicates radiolabeled nucleotides. b Analysis of the capping reactions prepared in a. Red and black arrowheads represent capped and uncapped RNA, respectively