FIGURE 4.

UTP-mediated nucleotide addition following U removal by the 3′-U-exo. PPi-containing reactions used purified editing complex and were much as in Figure 3 ▶ except the UTP was added after a delay, as indicated by the time points on the right, next to the “UTP”. Asterisks indicate the time that the reactions were terminated. (A) 3′-U-exo reaction. Precleaved U-deletion reaction containing no UTP was incubated for only 10 sec. (B–D) UTP addition after a delay. (B) Precleaved U-deletion reactions. (C) Precleaved U-deletion reactions instead using rapidly prepared cell lysate (0.15 μg; see Materials and Methods) and 0.5 mM PPi. (D) Reactions using intact substrate, with the initial incubation without UTP for 30 min to allow endonuclease as well as 3′-U-exo action; after such an incubation, the TUTase activity is somewhat decreased.