Determination of the molecular weights of RNAs by low-speed sedimentation equilibrium: 16 S ribosomal RNA as a model compound

Abstract

Problems associated with the determination of molecular weights of ribonucleic acids by sedimentation equilibrium have been investigated, using the sodium salt of 16 S ribosomal RNA from Escherichia coli as a model system, for which a chemical molecular weight has been computed from the sequence data (Fellner et al. , 1972; Fellner & Zimmermann, 1974) . It is shown that 16 S rRNA solutions exhibit significant interaction, probably in the form of a reversible dimerization. The sedimentation equilibrium procedure of Rowe & Rowe (1970 a,b ) is used to remove both interaction and non-ideality terms resulting in an estimated ideal molecular weight of 5·21 ± 0·11 × 10 5 for the monomeric species, based upon a partial specific volume &-v = 0·523 cm 3 ·g −1 determined using a Digital Density meter. This molecular weight value agrees well with the computed formula weight (5·33 ± 0·05 × 10 5 ), and implies that even for a polyelectrolyte such as a nucleic acid, &-v , measured conventionally, is a satisfactory parameter so far as hydrodynamic measurements are concerned. The partial specific volumes at 5°C of the sodium salts of poly(U), poly(A) and poly(U)·poly(A) have been measured and found to be closely similar (0·527, 0·531 and 0·529 cm 3 ·g −1 , respectively), and do not differ significantly from that of 16 S rRNA; suggesting that variation in base composition and secondary structure of RNAs has little effect on &-v , under defined conditions of temperature, ionic strength and counter-ion composition. A new method for measuring the absolute fringe displacement in the interference patterns obtained at sedimentation equilibrium has been developed and applied in this study (see Appendix).