Fig. 4.

Effect of cap 4 methylation on TbCe1 phosphotransferase activity. (A) Protein titration. The standard RNA kinase assay (10 µL) contained 100 µM [γ-³²P] ATP and 100 nM of either SL-pRNA, pRNAr1 (with 2′-O ribose methylation at position 1) or pRNAr1,2,3,4 (with 2′-O ribose methylation at position 1, 2, 3, and 4) and TbCe1, as indicated. The yield of ³²P-labeled RNA was plotted as a function of input TbCe1. (B) Kinetics. Standard RNA kinase assay (80 µL) contained 160 ng of TbCe1 and 100 nM of either SL-pRNA, pRNAr1, or pRNAr1,2,3,4. An aliquot (10 μL) was withdrawn at the indicated time point, and the products were resolved by denaturing PAGE. The yield of ³²P-labeled RNA product is plotted as a function of incubation time. (C) ATP titration. The standard RNA kinase assay (10 µL) contained 200 ng of TbCe1, 100 nM of either SL-pRNA, pRNAr1, or pRNAr1,2,3,4, and [γ-³²P] ATP at the indicated concentration. The yield of ³²P-labeled RNA was plotted as a function of ATP concentration. The data shown represent the average of three separate experiments. SE bars are included for each datum point.