To identify the region(s) of the TBSV RNA bound by the recombinant Ded1p, we performed electrophoresis mobility shift assay (EMSA) with purified components. Comparison of 32 Plabeled TBSV (+) and (2)RNAs in binding to purified recombinant Ded1p revealed that (2)RNA bound more readily to Ded1p in vitro than (+)RNA ( Figure 5B , lanes 2-6 versus 8-12). Additional EMSA experiments using the four regions in DI-72 (2)repRNA ( Figure 5A ) as unlabeled competitors revealed that RI(2) was the most efficient in outcompeting the 32 P-labeled TBSV (2)repRNA in binding to Ded1p ( Figure 5C , lanes [8] [9] . This is important since RI(2) is the 39 end of (2)repRNA and contains important cisacting elements, such as the promoter and a short enhancer sequence for (+)RNA synthesis [46, 47] .