FIGURE 4.

5′-Adenylation of P4–P6, a long and structured RNA substrate. The 160-nt RNA substrate was 5′-³²P-radiolabeled. Following 5′-adenylation (t = 0 h, 8 h, and 24 h using the optimal incubation conditions of Fig. 3), the 5′-terminal 14-nt portion of the RNA was cleaved using an 8–17 deoxyribozyme (see Materials and Methods), thereby allowing the 5′-adenylation to be detected by 20% PAGE. In all lanes, the band corresponding to the 146-nt 3′-portion of the P4–P6 RNA is present near the wells (not shown).