FIG. 5.

ATPase activity of UAP56. (A) ATPase activity of recombinant Flag-UAP56 was measured as described in Materials and Methods, with or without a 100-nt RNA derived from the β-globin mRNA sequence and/or recombinant Aly/REF. Pi, released phosphate; ori, origin of chromatography. (B) Quantitation of relative ATPase activities from four independent experiments as described for panel A. Averages and standard errors are shown. (C) ³⁵S-labeled Aly/REF protein produced in the E. coli T7 S30 system (Promega) was incubated with GST alone (GST; 300 pmol), GST-UAP56 (GST-UAP; 300 pmol), or GST-TAP231 (GST-TAP; 10 pmol) (12, 15) in the presence of RNase A and in the absence (lanes 2, 3, and 6) or presence (lanes 4, 5, 7, and 8) of recombinant TAP231 (10 and 100 pmol; lanes 4 and 5, respectively) or UAP56 (300 and 3,000 pmol; lanes 7 and 8, respectively), and GST pull-down was performed as described for Fig. 4B. ³⁵S-labeled Aly/REF protein was visualized by fluorography.