Characterization of the secondary structure features of Escherichia coli, Caldariella acidophila and mammalian ribosomal RNA species by chemical modification of sterically exposed bases

The helix content of rRNA species (Escherichia coli, Caldariella acidophila, rat liver) and the G · C content of their bihelical domains have been investigated by chemical modification of uracil and cytosine residues with probes specific for sterically exposed bases. By using radioactively labelled rRNA, G · C base pairs and the sum of A · U plus G · U base pairs have been quantified assuming that they are numerically identical with the unreactive cytosine and uracil rings, respectively. Exposed uracil bases were probed by their conversion to alkali-labile, non-ultraviolet-absorbing sulphonated adducts, with 1.33 M bisulfite pH 7, at 20°C; the adducts can be separated from unreacted uracil, and quantified, by cation-exchange chromatography of RNase T2 plus pancreatic RNase digests of bisulfite-modified rRNA. Exposed cytosines were probed by their conversion to methoxyaminated, alkali-stable, derivatives with 1 M methoxyamine, pH 5.5, at 37°C, and quantified by monitoring the CMP/AMP radioactivity ratio after alkaline hydrolysis of modified rRNA. Exposed uracil rings can also be estimated spectrophotometrically by the alkali-catalyzed reversal of the non-ultraviolet-absorbing sulphonated adducts after separation of the latter from unreacted uracil. The cytosine deamination reaction, catalyzed by bisulfite at pH 6, has also been investigated and found to exhibit little specificity for sterically exposed bases of rRNA, the (G + C)-richer rRNA species of C. acidophila being considerably less susceptible to non-specific deamination than the (G + C)-poorer rRNA of E. coli. A high degree of congruence is shown to exist between results obtained by chemical modification and melting hyperchromicity experiments.