1. Supplementary data 3: Steady state kinetic analysis of ApG-initiated RNA synthesis by the human E627K PB2 influenza A virus RNA polymerase complex at varying temperatures in the presence of NP. (A)ApG-initiated RNA synthesis was conducted using a 137-nt long template with the human Pol complex protein at 34, 37 and 42oC with varying concentrations of NTP substrates in the presence of NP. Sufficient NP was added to coat the 137-nt long template. The reactions were conducted as described in Figure 3 and the products were analyzed by 20% denaturing gels. C: No polymerase control. LC: loading control. (B, C and D) The reaction rates at each of temperatures and NTP concentration were normalized with the maximum rate (apparent Kcat=1/mole min) at 42oC at the highest NTP concentration of 500 mM, and the calculated relative reaction rates were plotted for the determination of Km values by the Michaelis-Menten equation. The PB2 concentration of the human E627K Pol complex was used for determining the apparent Kcatvalues. The data is summarized in Table 2.