Nuclease S1 analysis of eubacterial 5S rRNA secondary structure

Abstract

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 allel 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.