Figure 5. E.r. RT forms a dimer in solution in the absence and presence of D4A RNA.

(a) Molecular weight (MW) estimation by SV-AUC. Representative c(s_20,w) distributions for the E.r. RT (orange) and the E.r. RT–D4A complex (blue) were plotted against the sedimentation coefficient s_20,w (standardized to 20 °C and water) (left panel). In two independent experiments for the RT domain, the fitted f/f₀ values were both 1.29, whereas the peak s_20,w values were 4.1 s and 4.2 s, yielding estimated MW values of 61 kDa and 62 kDa, respectively. From the crystal structure of the E.r. RT dimer, the predicted f/f₀ is 1.22 and the predicted s_20,w is 4.7 s (US-SUMO⁴⁷), which are in good agreement with experimentally determined values. In both of the two independent experiments for RT–D4A complex, the fitted f/f₀ value was 1.52 and the peak s_20,w value was 7.4 s, yielding an estimated MW of 116 kDa. (b,c) MW analysis using SEC-MALS. The experiments for E.r. RT (panel b, shades of orange) and E.r. RT–D4A complex (panel c, shades of blue) were performed over a range of concentrations (in mg/mL), and the MW at each concentration was plotted as squares, triangles and circles respectively (upper right legend) on the right y-axis. For each concentration, the UV trace (curve) was plotted on the left y-axis with the elution volume indicated on the x-axis. The corresponding plot for the E.r. D4A RNA alone is provided as Supplementary Fig. 4d. For RT domain, the MW at the elution peak was 63 kDa at 0.01 mg/mL, 67 kDa at 0.08 mg/mL and 66 kDa at 0.01 mg/mL. For RT–D4A complex, the MW at the elution peak was 104 kDa at 0.02 mg/mL, 110 kDa at 0.08 mg/mL and 112 kDa at 0.01 mg/mL.