FIGURE 10.

Experiments to confirm the 5′-AppRNA structure and its mechanism of formation. (A) Reaction of 5′-³²P-radiolabeled 34-mer RNA substrate S9 with T4 RNA ligase in the absence of ATP and in the absence or presence of excess unradiolabeled 5′-AppRNA derived from 17-mer RNA substrate S4. The asterisk on *S9 denotes a 5′-radiolabel. As expected, only when unradiolabeled 5′-App-S4 is included is a higher molecular weight radiolabeled ligation product formed (*S9 + S4). For each set of lanes, the three time points are at 0.5 min, 10 min, and 1 h (12% PAGE). An appropriate blocking oligo was included in excess for both of the RNA substrates (see Materials and Methods). (B) 5′-adenylation of 17-mer RNA substrate S1 using either 5′-³²P-S1, α-³²P-ATP, or both. The +5/0 blocking oligo promotes formation of all three side products (see Fig. 3 ▶), therefore providing a stringent survey of the products into which the radiolabel becomes incorporated. The band patterns are as expected for the 5′-adenylation mechanism of Figure 1B ▶. For all sets of lanes, the four time points are at 0.5 min, 10 min, 1 h, and 6 h (20% PAGE). The three sets of lanes are numbered at the bottom for reference (see text).