Effects of alanine mutations on RNA phosphorylase activity. (A) Reaction mixtures (10 µL) containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.5 mM (NH4)2PO4, 1 pmol (0.1 µM) of 5′ 32P-labeled 24-mer RNA (depicted at bottom with the 5′ label denoted by a dot), and 1 pmol (0.1 µM) wild-type or mutant recombinant PNPase homotrimer as specified above the lanes were incubated for 15 min at 37°C. The reaction products were analyzed by urea-PAGE and visualized by autoradiography. (B–D) Reaction mixtures (70 µL) containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.5 mM (NH4)2PO4, 0.1 µM 5′ 32P-labeled 24-mer RNA, and 0.1 µM wild-type or mutant PNPase homotrimer as specified above the gels were incubated at 37°C. Aliquots (10 µL) were removed at the times specified above the lanes and quenched immediately with formamide/EDTA. The reaction products were analyzed by urea-PAGE and visualized by autoradiography. The positions of the input 24-mer substrate and a 5′ 32P-labeled trinucleotide reaction product are indicated at the left of each gel.
