Nuclease S1 analysis of eubacterial 5S rRNA secondary structure.

Single-strand-specific nuclease S1 was employed as a structural probe to confirm locations of unpaired nucleotide bases in 5S rRNAs purified from prokaryotic species of rRNA superfamily I. Limited nuclease S1 digests of 3'- and 5'-end-labeled [32P]5S rRNAs were electrophoresed in parallel with reference endoribonuclease digests on thin sequencing gels. Nuclease S1 primary hydrolysis patterns were comparable for 5S rRNAs prepared from all 11 species examined in this study. The locations of base-paired regions determined by enzymatic analysis corroborate the general features of the proposed universal five-helix model for prokaryotic 5S rRNA, although the results of this study suggest a significant difference between prokaryotic and eukaryotic 5S rRNAs in the evolution of helix IV. Furthermore, the extent of base-pairing predicted by helix IV needs to be reevaluated for eubacterial species. Clipping patterns in helices II and IV appear to be consistent with a secondary structural model that undergoes a conformational rearrangement between two (or more) structures. Primary clipping patterns in the helix II region, obtained by S1 analysis, may provide useful information concerning the tertiary structure of the 5S rRNA molecule.