Analytical ultracentrifugation of the RSE RNA. (A) Sedimentation equilibrium data were collected at three rotor speeds (9,000, 10,500, and 12,000 rpm) at 20°C with a loading concentration of 20 μg/ml monitored at 260 nm. Data plots were offset by 0.2 absorbance units (AU) for clarity. The solid line represents the predicted mobility of an RNA with a molecular mass of 94,173 Da (the mass calculated from the RSE RNA sequence) as determined by the Ultrascan software. (B) Comparison of curve fits to the 12,000-rpm data set from panel A. Using the Ultrascan software, the molecular mass was constrained to either the monomeric (solid line; 94,173 Da) or dimeric (dashed line; 188,346 Da) calculated mass, and both of these predicted curves were overlaid on the graph. (C) Van Holde-Weischet analysis of the velocity sedimentation data at 35,000 rpm and 20°C shows that the sample is primarily a single species. This plot was constructed by analyzing 50 equally spaced boundary fractions, spanning 10 to 90% of the boundary. The average S value determined was 7.4. (Inset) The same data analyzed using the time derivative method produced the same S value. MW, molecular weight; Sed Coeff, sedimentation coefficient.