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Figure 4 | Virology Journal

Figure 4

From: Biochemical characterization of the fidelity of poliovirus RNA-dependent RNA polymerase

Figure 4

Traps to reduce multiple turnovers. (A) Titration of heparin inhibition of 3Dpol. 1 μM Sym/Sub U was incubated with 500 μM ATP, 1 μM 3Dpol and increasing amounts of heparin. Polymerase activity was assessed in 20% PAGE, as described in Methods. The data were fit to a single exponential curve for inhibition. (B) Apparent single turnover kinetics for 3Dpol with Sym/Sub U and incorrect base U in the presence heparin trap. 1 μM 3Dpol and 1 μM Sym/Sub U were incubated with 750 (squares) or 1500 μM UTP (circles) for the indicated time (in minutes) as described in Methods, except that 100 nM heparin was added immediately after reaction initiation. Data (from all products) were plotted as a function of time in seconds and fit to a single exponential (Table 2). (C) Apparent single turnover kinetics for 3Dpol with Sym/Sub U with incorrect base U in the presence of cold trap. 1 μM 3Dpol and 1 μM Sym/Sub U were incubated with 750 (squares) or 1500 μM UTP (circles) for the indicated time (in minutes) as described in Methods, except that ten-fold molar excess unlabeled Sym/Sub U was added immediately after reaction initiation. Data (from all products) were plotted as a function of time in seconds and fit to a single exponential (Table 2).

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