Fig. 3. Mapping the location of the replication silencer using DNA oligonuclotides in an in vitro tombusvirus RdRp assay. (A) Schematic representation of the RIV template, which includes only RIV sequences (Figure 1A). (B) The actual sequence of SL3 in construct RIV is shown on the top, and the complementary ssDNAs are shown below the target sequence. The critical nucleotides in SL3 are boxed. The position of the last nucleotide shown is indicated. A representative denaturing gel analysis of radiolabeled RNA products synthesized by in vitro transcription with CNV RdRp is also shown. Each RdRp reaction contained the same amounts of the RIV template and the ssDNA competitor indicated. The lane marked ‘–’ represents the control, with no ssDNA competitor added. Asterisks depict the RdRp products generated by de novo initiation from the 3′-terminus. The band migrating below the template-sized band (marked with asterisk) is the result of internal initiation, probably at an internal cytidylate (position 6 from the 3′ end). The molecular marker (the RIV construct) is shown as ‘tr’ on the left. See Figure 1 for further details. (C) The actual sequence of gPR is shown on the top and the complementary ssDNAs are shown below the target sequence. The critical sequence in gPR is boxed. See (A) for further details.