To test the in vitro activity of Tef4p, different concentrations (26 and 13 pmol) of purified FLAG/His 6 -Tef4p was added to 0.25 mg (4 pmol) DI-72 (+)repRNA transcript and incubated in the presence of yeast cell-free extract and reaction buffer for 10 minutes at RT followed by the addition of MBP-p33 and MBP-p92 along with the rest of the reaction components. The reaction was performed at 25uC for 3 h and analyzed as above.