Figure 2.

Integrity of the leader–reporter RNA complexes after incubation in translation mixtures. (A) The integrity of leader–reporter RNA complexes after incubation in translation mixtures (RRLs) on ice (lanes 1–2 and 5–6) or at 30°C for 15 min (lanes 3–4 and 7–8) was monitored on an 8% (w/v) non-denaturing acrylamide gel. In some mixtures, 0.2 mM cap analogue was added to translation mixture to block translation (lanes 5–8). The ratio of annealed-to-free reporter G-LEAD_C, after incubation at 0°C, in mock- or cap analogue-treated RRL, was set to unity, to permit comparisons (lanes 1–4 and lanes 5–8, respectively). (B) Inhibition of helicase in RRL stabilizes leader–reporter RNA complex. RNA integrity was monitored in the absence (lanes 1–4) or presence (lanes 5–6) of 2 mM AMP-PNP that inhibits ATP-dependent helicase activities. (C) Putative covalent bond formation between leader and reporter RNAs. To test whether translational enhancement by capped leader RNA was mediated by putative covalent bond formation between leader and reporter RNAs, leader (G-LEAD_C and G-LEAD_R) and reporter RNAs were incubated in translation mixtures at 30°C (lanes 4–6 and 9–10) or on ice (lanes 1–3 and 7–8) for 15 min. The RNAs in RRLs were extracted and then resolved on an 8% (w/v) non-denaturing acrylamide gel with (lanes 7–10) or without (lanes 1–6) heat denaturation at 95°C for 5 min.