Fig. 2.

The synthesis of the (³³P, 1-²H) dual-labeled ribozyme-PRPP species. (a) Ribose isotopically substituted with 1-²H was converted first to ribose 5-phosphate and then PRPP by using ribokinase and PRPP synthase successively in the presence of ATP. (b) PRPP purified by using a DEAE column was reacted with adenosine-5′-phosphorimidazole (19) to synthesize adenylylated PRPP (AppRpp), which was purified by reverse-phase HPLC. (c) Ribozyme (Rz) radiolabeled during T7 transcription with ³³P was covalently attached at its 3′ end to the isotopically modified PRPP by using T4 RNA ligase.