FIGURE 1.

Thermal stability of the replicase. A, time course of RNA replication by replicase purified by the standard method and the effects of incubation before the reaction. The replicase (200 nm) was incubated at 37 °C in the absence of RNA and NTPs for the indicated times (0, 0.5, 2, and 4 min) before the replication assay, and then RNA replication was measured at 37 °C after the addition of 50 nm template RNA (s130) and NTPs. The label “no rep” indicates the results of a control experiment without replicase. B, effects of preincubation on residual replication activity. Each form of replicase was incubated at 37 °C in the absence of template RNA and NTPs before the replication assay for the indicated times. The RNA replication reaction was measured as described in Fig. 1A, and the replication rates were determined by linear regression analysis. The replicase purified by the standard method is indicated by the filled circles. Replicase prepared by dialyzing the standard replicase with 10 mm DTT (U-form) is indicated by circles. Replicase prepared by heat inactivation of the standard replicase followed by gel-filtration chromatography (S-form) is indicated by triangles. The data for the S- and U-forms were fitted to a single exponential decay curve as eq. 1 (gray lines). C, effects of preincubation temperature on residual activity. U-form replicase was incubated at different temperatures before the replication reaction was carried out for 2 min in the absence of RNA and NTPs, and then RNA replication rates of the replicase were measured as described in Fig. 1A.