FIG. 3.
PPi-dependent rescue of chain-terminated RNA synthesis. (A) Principle of the reaction. The inclusion of obligate chain terminators in the GG-primed reaction yields various abortive products. The subsequent addition of PPi provides conditions that allow excision of incorporated 3′-dNMPs and, in turn, rescue of RNA synthesis. (B) RNA synthesis was allowed to proceed for 30 min in the presence of a 3 μM concentration of each NTP and 10 μM 3′-dGTP, 3′-dUTP, 3′-dCTP, or 30 μM 3′-dATP (lanes 0). Rescue of RNA synthesis was then monitored over another 30 min through the addition of increasing concentrations of PPi. Lanes 1 to 6, 31, 62, 125, 250, 500, and 1,000 μM PPi. Arrows point to rescued primers. Controls in lane C show reactions in the absence of obligate chain terminators.