FIG. 6.

Pta1 is required for both cleavage and poly(A) addition of GAL7 pre-mRNA substrate. Equal amounts of protein were mixed with ³²P-labeled GAL7 substrate and incubated at 30°C for 20 min as described in Materials and Methods. Products were resolved on denaturing polyacrylamide gels and visualized by autoradiography. (A) Processing assay with full-length GAL7-1 RNA substrate and ATP. (B) Cleavage assay in which ATP is replaced with 2′-dATP to block the poly(A) addition step. (C) Poly(A) addition assay with precleaved RNA substrate GAL7-9. Lanes 1, precursor; lanes 2, wild-type (WT) extract; lanes 3, pta1-1 mutant extract; lanes 4, pta1-2 mutant extract; lanes 5, pta1-1 extract supplemented with CF II; lanes 6, pta1-2 extract supplemented with CF II; lanes 7, reaction with CF II only. The positions of the full-length GAL7-1 precursor RNA, poly(A)⁺ RNA, upstream and downstream cleavage products, and precleaved GAL7-9 precursor (top to bottom, respectively) are indicated by the bars on the left of each panel.