Initiation of RNA synthesis by Δ21 using linear and circular RNAs. (A) Requirement for GTP for RNA synthesis by Δ21. The RdRp reactions using +1C-L and +1C-C were separated on a gel and the 13-nt RNA product for +1C-L and ~65-nt product for +1C-C were quantified and plotted against different concentrations of GTP. The amount of product formed with 200 μM GTP was considered as 100%. (B) Schematic showing the RNA synthesis initiation sites on +1C RNA. Arrows correspond to the possible initiation sites for the nucleotides analogs given on the left. The RNA sequence is given in the 3′-to-5′ direction, and the line underneath shows the bond formed during circularization of the RNA. (C) Analysis with GTP analogs. A representative gel picture showing the RNA synthesis products obtained by using different GTP analogs with templates +1C-L (left) and +1C-C (right). The analogs used are given on the top, and the position of 13-nt product is shown at the left of the gel. All reactions were supplemented with 1 μM GTP, which is needed for elongative RNA synthesis. (D) Analysis of LE21-L and LE21-C using GTP analogs. Since LE21 has only one C (at the 3′-end of linear RNA), the analogs used can initiate RNA synthesis only from one position. The analogs used are given above and the sizes of RNA products to the left of the gel. The bottom of the gel shows the quantitative analysis of the 21-, 42-, and 63-nt products obtained with respective analogs. For LE21-L, the amount of 21-nt product generated with GTP was considered as 100%, and for LE21-C, 100% is the amount of ~60-nt product generated with GTP. All the values are after normalizing for the number of radiolabeled CMPs incorporated.
