FIG. 4.

Effect of temperature on the RdRp activity of recombinant p88 and p88C and the plant TCV RdRp. (A) Representative denaturing gel analyses of radiolabeled RNA products synthesized by in vitro transcription with p88, p88C, and the plant TCV RdRp using satC(−) as the template. Products are marked as shown in the legend to Fig. 3. The exposure time was different for each RdRp assay due to the increased activity for p88C. (B) The relative amounts of M and D RdRp products were measured at various temperatures, as shown. The amount of product D obtained at 25°C was selected as 100% separately for each RdRp preparation. Note that the increased level of product D at 30 and 37°C is due to terminal transferase activity present only in the p88 preparation, as confirmed by S1 nuclease digestion (not shown). The terminal transferase activity was not significant at 25°C (not shown). Terminal transferase activity in the p88 preparation was confirmed by obtaining template-sized product (similar in size to product D) when the RdRp reaction contained [³²P]UTP in the absence of the other three nucleotides. This terminally labeled product, unlike product D, was fully S1 nuclease sensitive (not shown). Product E was not included because its amount varied in separate experiments.