Enhanced activity of the affinity-purified tombusvirus replicase assembled in CFE from <i>pah1Δ nem1Δ</i> yeast.

<p>(A) Scheme of the tombusvirus replicase assay. CFEs were prepared from wt and <i>pah1Δ nem1Δ</i> yeasts, followed by the addition of purified recombinant tombusvirus p33 and p92^pol replication proteins and DI-72 (+)repRNA in the presence of rATP and rGTP. The <i>in vitro</i> assays were programmed with RI/III (−)RNA in the presence of rATP/rCTP/rGTP and ³²P-rUTP. (B) Top panel: Representative denaturing gel of ³²P-labeled RNA products synthesized by the purified tombusvirus replicase <i>in vitro</i>. Each experiment was repeated. Bottom panel: Western blot analysis of p33 in the shown replicase samples.