- RNase M5 of Bacillus subtilis specifically cleaves a 179-nucleotide precursor 5S rRNA to yield mature 5S rRNA (116 nucleotides) and two fragments derived from the termini. Possible recognition elements for RNase M5 within the precursor structure include nucleotide sequences arranged with 2-fold rotational and translational symmetry about the substrate bonds. We have used bacteriophage T4 RNA ligase to construct, from synthetic oligonucleotides and mature or precursor 5S rRNA fragments, test substrates lacking these symmetry elements. The susceptibilities of the artificial substrates to RNase M5 demonstrate that the symmetrically arranged sequences are not used in the RNase M5 interaction with the precursor. Additionally, the synthetic protocols permitted the invention of an acid-soluble assay for RNase M5 and, potentially, other specific endoribonucleases.