Purification, cloning, and characterization of the 16 S RNA m(2)G1201 methyltransferase from Escherichia coli

The methyltransferase that forms m(2)G1207 in Escherichia coli small subuni t rRNA has been purified, cloned, and characterized. The gene was identifie d from the N-terminal sequence of the purified enzyme as the open reading f rame yjjT (SWISS-PROT accession number P39406), The gene, here renamed rsmC in view of its newly established function, codes for a 343-amino acid prot ein that has homologs in prokaryotes, Archaea, and possibly also in lower e ukaryotes. The enzyme reacted well with 30 S subunits reconstituted from 16 S RNA transcripts and 30 S proteins but was almost inactive with the corre sponding free RNA. By hybridization and protection of appropriate segments of 16 S RNA that had been extracted from 30 S subunits methylated by the en zyme, it was shown that of the three naturally occurring m(2)G residues, on ly m2G1207 was formed. Whereas close to unit stoichiometry of methylation c ould be achieved at 0.9 mn Mg2+, both 2 mM EDTA and 6 mM Mg2+ markedly redu ced the level of methylation, suggesting that the optimal substrate may be a ribonucleoprotein particle less structured than a 30 S ribosome but more so than free RNA.