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. 2005 Jul 19;102(30):10460–10465. doi: 10.1073/pnas.0504249102

Fig. 4.

Fig. 4.

Pateamine stimulates eIF4AI helicase activity. (A) Reactions were performed with RNA duplexes RNA-1/RNA-11 or RNA-1/RNA-12 for 15 min at 35°C in the presence of 0.36 μg of recombinant protein. The presence or absence of 1 mM ATP or 10μM pateamine is indicated. Reactions were resolved on 12% native gels, which were dried, and exposed to x-ray film (Kodak) at -80°C for 12 h with an intensifying screen. The position of migration of duplex or radioactive single-strand RNA is indicated to the left. (B) Helicase assays performed with recombinant Ded1p protein in the presence of pateamine. Conditions were similar to those described for eIF4AI. (C) In vitro splicing reactions in the presence of pateamine. In vitro splicing reactions were performed with the AdML pre-mRNA and analyzed, as described (17). Reaction products were separated on a 15% polyacrylamide/8 M urea gel, which was dried, and exposed to X-Omat (Kodak) x-ray film at -80°C for 1 h. The position of migration of the pre-mRNA (lane 1) and spliced mRNA (lane 2) is indicated to the right. Splicing reactions were performed without pateamine (lane 3), in the presence of increasing concentrations of pateamine [lane 4 (0.5 μM), lane 5, (2 μM), and lane 6 (10 μM)], or in the absence of exogenously added ATP (lane 7).