FIGURE 6.

Mutational effects on the kinetics of 2′-PO₄ removal. Reaction mixtures containing 100 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM NAD⁺, 0.2 µM 5′ ³²P-labeled 6-mer 2′-PO₄ RNA, and 0.5 µM RslTpt1 (wild-type or mutant as specified) were incubated at 37°C and quenched at the times specified (above the lanes) by addition of 3 volumes of cold 90% formamide, 50 mM EDTA. The reaction products were analyzed urea-PAGE and visualized by autoradiography. The inset box depicts the 6-mer 2′-PO₄ RNA substrate, 2′-P-ADPR RNA intermediate, and 2′-OH RNA product of the two-step Tpt1 reaction.