Characterization of Pla1 poly(A) polymerase activity. (A) Time course of primer extension. A reaction mixture (85 µL) containing 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM MnCl2, 0.5 mM DTT, 1 mM ATP, 0.1 µM 5′-32P labeled RNA primer, and 0.04 µM Pla1 were incubated at 30°C. At times specified, aliquots (10 µL) were withdrawn and quenched immediately by adjustment to 47.5% formamide/12.5 mM EDTA. (B) Divalent cation specificity. Reaction mixtures (10 µL) containing 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM ATP, 0.1 µM (1 pmol) 5′-32P labeled RNA primer, and 0.04 µM Pla1 (400 fmol), and either no metal (−) or 1 mM of the indicated divalent cation (as the chloride salt or CdSO4) were incubated at 30°C for 30 min and subsequently quenched. (C) Metal mixing. Reaction mixtures (10 µL) containing 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.5 mM MnCl2, 1 mM ATP, 0.1 µM (1 pmol) 5′-32P labeled RNA primer, either no enzyme (−E) or 0.04 µM Pla1 (400 fmol), and either no other metal (−) or 0.5 mM of the indicated divalent cation (as the chloride salt or CdSO4) were incubated at 30°C for 30 min and subsequently quenched. (D) NTP specificity. Reaction mixtures (10 µL) containing 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM MnCl2, 0.5 mM DTT, 0.1 µM 5′-32P labeled RNA primer, and 0.04 µM Pla1, and either no added NTP (−) or 1 mM ATP (A), GTP (G), CTP (C), UTP (U), or dATP (dA) as specified were incubated at 30°C for 30 min and subsequently quenched. Primer extension products for all assays shown in Figure 3 were resolved by 8% urea-PAGE and visualized by autoradiography. The size in nucleotides of a DNA ladder (MspI digest of pBR322 plasmid) is indicated on the right of each panel. The proportion of the primer extended by 1, 2, or 3 nt in D was quantified in ImageQuant-TL after scanning the gel with a Typhoon FLA7000 imager.
