FIG. 7.
Incorporation of RMP into sym/sub-C and sym/sub-U by wild-type 3D(M) and mutant 3D(M-M296I). (A) Denaturing PAGE of the 32P-labeled products of the polymerase-catalyzed RMP incorporation into sym/sub-C. 3D (3 μM) was mixed with sym/sub-C RNA (0.5 μM duplex) for 10 min for annealing, and then RTP (50 μM) was added to the reaction mixture. Reactions were stopped at the indicated times by the addition of EDTA. (B) Kinetics (0, 20, 120, 300, and 1,200 s) of RMP incorporation into sym/sub-C by 3D(M) (○) and 3D(M-M296I) (•). Shown are averages of results from three experiments performed like that whose results are shown in panel A. Standard deviations of densitometry values are given. (C and D) Experiments were performed and symbols are the same as those described for panels A and B, respectively, except that sym/sub-U was used as the template-primer. Procedures are detailed in Materials and Methods.