FIG. 1.

Template switching by the recombinant TCV RdRp in vitro. (A) Sequences and structures of RNA templates used in the TCV RdRp reactions. Construct Mot1/pr contains the minus-stranded motif1 hairpin replication enhancer (shown as the hairpin structure [39]) and the 3′-terminal plus-strand initiation sequence (boxed with dotted line) of TCV satC. Construct R3(−) contains the minus-stranded replication enhancer RIII(−) (49) and the minus-stranded 3′-terminal cPR11 promoter of the related TBSV (boxed) (47). Construct AU1 contains an artificial AU-rich sequence (encircled in gray box) (10), while the same-sized construct GC1 carries an artificial GC-rich sequence (shaded area) (10). In addition, constructs AU1 and GC1 contain the same 5′ and 3′ sequences (cPR11 is boxed); thus, these constructs are different only in the shaded regions. Note that the GC and AU regions differ not only in their sequences but also in the strength of the hairpin structures that could potentially influence initiation and/or recombination. (B) Detection of recombinants in an in vitro TCV RdRp assay. RNA templates were used in equal amounts (15 pmol/reaction mixture) under the conditions described in Materials and Methods. The RdRp products synthesized by in vitro transcription with the recombinant TCV RdRp were analyzed in a fully denaturing gel. The names of the RNA constructs used in the TCV RdRp reactions are shown above the lanes. The positions of heterorecombinants are marked with asterisks. Note that the combination of R3(−) and Mot1/pr resulted in two heterorecombinants (double band). The homorecombinants derived from the Mot1/pr template are also marked. Note that under the conditions used (reduced nucleotide concentration), the recombinant TCV RdRp performs 3′-terminal (primer) extensions for all four templates at various efficiencies. (C) TCV RdRp products obtained with the Mot1/pr template. The molecular size markers, which were obtained with T7 transcription, are shown on the left.