A Co-Opted DEAD-Box RNA Helicase Enhances Tombusvirus Plus-Strand Synthesis
Figure 5
Ded1p binds to the 3′ end of the TBSV (−)RNA.
(A) Schematic representation of the four regions in DI-72 repRNA used in the binding assay. (B) In vitro binding assay with purified Ded1p using 32P-labeled ssRNA templates. The assay contained the (−) or (+) DI-72 repRNA (∼0.1 pmol) plus increasing amount [0.4 µg (lanes 2–3 and 8–9), 0.8 µg (lanes 4–5 and 10–11) and 1.2 µg (lanes 6 and 12)] of purified recombinant Ded1p. The free or Ded1p-bound ssRNA was separated on nondenaturing 5% acrylamide gels. (C) RNA gel shift analysis shows that Ded1p binds the most efficiently to RI(−). 32P-labeled DI-72 (−)repRNA template (∼0.1 pmol) and unlabeled competitor RNAs (3 and 6 pmol) representing one of the four regions (see panel A) were used in the competition assay. The Ded1p - 32P-labeled ssRNA complex was visualized on nondenaturing 5% acrylamide gels. Each experiment was repeated at least three times.