Substrate specificity of yPAP toward various purine triphosphate analogues (A) Elongation of RNA primer 5′ UGU GCC CGA 3′ by yPAP. A mixture containing 200 nM 5′- 32 P-radiolabeled RNA primer, 4 U/μL yPAP, 250 μM analogue triphosphate, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl 2 , 0.2 mM EDTA, 100 μg/ml acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1 , radiolabeled 10-bp DNA ladder; lane 2 , 5′- 32 P-radiolabeled unextended primer (no NTP); lane 3 , <t>ATP;</t> lane 4 , 2′-dATP; lane 5 , 3′-dATP; lane 6 , 2-Cl-ATP; lane 7 , 2-Cl-dATP; lane 8 , ara-ATP; lane 9 , F-ara-ATP; lane 10 , Cl-F-ara-ATP; lane 11 , Cl-F-dATP, lane 12 , GTP; lane 13 , <t>dGTP,</t> lane 14 , ara-GTP. (B) Graphical representation of RNA primer extension by yPAP with various modified triphosphates. Shown is the distribution of single extension products (white) and full extension products beyond first incorporation (hatched) as a percentage of the total counts in each lane, as determined using ImageQuant software. These experiments were conducted in triplicate with similar results.
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