The helix content of rRNA species (Escherichiu coli, Calduriellu ucidophila, 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. nonultraviolet-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, alkalistable, 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. ucidophila 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.The principal secondary structure features of RNA species can be evaluated spectrophotometrically by melting hyperchromicity methods 11-31. The amount of bihelical secondary structure (i.e. the fraction of residues involved in hydrogen-bonded base-pairing) can be estimated by comparing the hypochromism of the partly double-helical polynucleotide to that of an appropriate fully bihelical analogue to denaturation from unpaired but stacked bases; solvents used to discriminate single-stranded stacking from helix to coil transitions, such as guanidinium chloride [7-91, may Ahhreviarrons. S-rRNA and L-rRNA indicate the RNA species isolated from the smaller and the larger ribosomal subunit, respectively: U* and U . SO3 are used, as convenient. to designate 5.6-dihydrouracil 6-sulphonate: C*, rnethoxyaminated cytosine.Enzymes. Pancreatic RNase (EC 3.1.27.5); T2 KNase (EC 3.1.27.1).also cause denaturation of bihelical segments to an extent depending on both solvent concentration and bihelix stability (Cammarano et al., unpublished work); this may constitute a source of error in investigating eukaryotic mRNA species, whose long poly(A) tails are fully stacked in neutral salt solutions; (d) unless specially built devices are used [lo], the potential of melting hyperchromicity methods is often limited by the availability of m...
