Mutational effects on poly(A) polymerase activity. Reaction mixtures (10 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 (1 pmol) 5′-32P labeled RNA primer, and the indicated amounts of WT, Y86F, Y86A, and Y86L Pla1 (A) or mutants Y86D and D153A (B) were incubated at 30°C for 30 min and subsequently quenched by adjustment to 47.5% formamide/12.5 mM EDTA. Primer extension products were resolved by 8% urea-PAGE and visualized by autoradiography. The size in nucleotides of a DNA ladder is indicated. In B, the proportion of the extended primer, either by 1 nt or >1 nt, was quantified in ImageQuant-TL after scanning the gel with a Typhoon FLA7000 imager.
