FIG. 2.

pan and pab1 mutant yeast extracts polyadenylate RNA substrates to aberrantly long lengths in vitro. The six 3′-end processing extracts (10 μg of total protein) described in the legend to Fig. 1B and a wild-type extract (YAS306) immunoneutralized for Pab1p (lanes 15 and 16) were programmed with CYC1 pre-RNA as described in Materials and Methods. Reaction products were visualized on a 6% polyacrylamide gel and visualized by autoradiography. Products of the cleavage reaction only (C) or of cleavage and polyadenylation of the CYC1 pre-RNA (A) are shown. Lane 1; DNA markers (M); lane 2, input CYC1 pre-RNA (precursor [pre]). The migration positions of the 5′-upstream cleavage product and the polyadenylated products are indicated to the right, and the sizes (in nucleotides) of the DNA markers are indicated to the left. WT, wild type; α-Pab1p, anti-Pab1p.