Fig. 3.

Analysis of the products of in vitro RdRp extension reactions of radioactive sg3a-384 RNA on the RW (−) RNA3 template. The products were separated in denaturing 1.2% agarose gels. Lane 1, mixture of in vitro-transcribed radioactive wt BMV RNA3, sgRNA4, and sg3a-384 RNA as size standards; lanes 2 and 3, primer extension reaction with a single (lane 3) or double (lane 2) amount of the radioactive sg3a-384 RNA. In addition, lanes 2 and 3 contain ³²P-labeled sgRNA4 as an internal loading standard that was added after the reaction. The reaction in lane 2 contains a double amount of the sg3a-384 primer. Lanes 4 and 5, control extension reactions of sg3a-384 primer on the BMV RNA2 template (lane 4) or without an RNA2 template (lane 5). Lanes 6 and 7, digestion of sg3a-384 extension products without (lane 6) or with (lane 7) S1 nuclease. Both, sgRNA4 (added post-reaction) and ss sg3a-384 RNA disappeared after S1 nuclease treatment (lane 7). Lanes 8 and 9, analysis of TNT activity of BMV RdRp, with the extension reactions completed with (lane 8) or without (lane 9) GTP. Both the RW(−) RNA3 template and the corresponding sg3a-384 RNA primer (* symbolizes the radioactivity) are shown schematically below (see Fig. 1 for more details).