Effect of the 3’-terminal sequence of template for the initiation efficiency of RNA replication. (A ) The sequence of Temp (GGG-UUC), Temp (GGG-UCC), Temp (GGG-CCC) and Temp (GGG-CCCC). (B) and (C) NV3Dpol (10 pmol) and each RNA templates (10 pmol) were incubated at 30°C and sampled at 0, 30, 60, 120 min (reaction volume = 40 μL). Aliquots were analyzed on an 8 M urea denaturing 10% PAGE and imaged with Fx imager after SYBRgreenII staining. Color-coded images were shown at the bottom. M; 10 bp DNA step ladder marker (Promega). Ref.; Temp (GGG-CCC) RNA. (D) and (E) Quantification of the amount of RNA strands from the denaturing PAGE (B) and (C), respectively. RNA quantification method was same as in Figure 3. Circle; Temp(GGG-UUC), triangle; Temp (GGG-UCC), square; Temp (GGG-CCC) and diamond; Temp (GGG-CCCC), respectively.