Supplementary data 2: Steady state kinetic analysis of ApG-initiated RNA synthesis by the avian E627 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 avian Pol complex protein at 34, 37 and 42oC with varying concentrations of NTP substrates in the presence of NP (see Experimental Procedures). The reactions were conducted as described in Figure 2. Sufficient NP was added to coat the 137-nt long template. 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 avian E627 Pol complex was used for determining the apparent Kcat values. The data is summarized in Table 2. At least three independent reactions were conducted for the analysis.