FIGURE 5.

TRAMP enhancement of the nuclease activity of Rrp6 does not require ATP or the presence of a poly(A) tail. A, GST-Rrp6 (0.3 pmol) was incubated with or without TRAMP (0.3 pmol in a 10-μl reaction in the presence of 3 fmol of 5′ [³²P]RNA substrate and either buffer, ATP (500 μm), or 3′-dATP (500 μm) for 60 min at 30 °C and analyzed as described in the legend of Fig. 3. The values listed below the lane numbers represent the average ± S.E. for the amount of substrate remaining after incubation and are calculated from the results of two independent experiments. B, 5′ [³²P]RNA substrate was preincubated with TRAMP in the presence (upper panel) or the absence (lower panel) of ATP. The reactions were phenol-extracted to remove TRAMP, and the RNA substrate was concentrated by ethanol precipitation. The RNAs were then incubated in the presence of GST-Rrp6 (1 pmol) for 60 min and analyzed as described in the legend of Fig. 3. C, graphic display of the rate of product formation by GST-Rrp6 for the poly(A)⁺ and poly(A)⁻ substrates in the experiments shown in B. Product was defined as RNAs shorter than the substrate and was quantified by storage phosphorimaging analysis.