Recognition of local nucleotide conformation in contrast to sequence by a rRNA processing endonuclease.

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Abstract
RNase M5 of Bacillus subtilis cleaves twice in a double-helical region of a 179-nucleotide precursor of 5S rRNA to yield mature 5S rRNA (116 nucleotides) plus fragments (21 and 42 nucleotides) derived from both termini. Previous experiments showed that the major recognition elements for the highly specific RNase M5 are in the mature domain of the precursor. One precursor residue, a G adjacent to the 5'' cleavage site, significantly enhances the rate of its own cleavage and that of the 3'' precursor fragment, so it must be an important component of the features recognized by the enzyme. This G residue is opposed in the helical substrate region to a C residue, which is at the 3'' terminus of the mature domain, presenting the question of whether RNase M5 specifically contacts the cleavage site on the basis of nucleotide sequence (the G residue per se) or on the basis of more general aspects of helical conformation. These alternatives were tested by fabricating partially synthetic test substrates for RNase M5. Experiments were performed on 5'' and 3'' half-molecules derived from mature 5S rRNA. The 3''-terminal C was removed by periodate oxidation and .beta. elimination and replaced in a [phage] T4 RNA ligase condensation with each of the 4 mononucleoside bisphosphates. Artificial precursor segments containing each of the 4 nucleotides adjacent to the 5'' cleavage site were added to the 5'' terminus of the 5S rRNA half-molecule. The modified half-molecules were then annealed to yield test substrates containing all permutations of complementary in contrast to noncomplementary nucleotides at the cleavage site. The susceptibilities of these test substrates show that conformation, not sequence, is the important feature in the locale of the cleaved bonds.