ERME METHYLTRANSFERASE RECOGNITION ELEMENTS IN RNA SUBSTRATES

Dimethylation by Erm methyltransferases at the N-6 position of adenine 2058 (A2058, Escherichia coli numbering) in domain V of bacterial 23 S rRNA confers resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics. The ErmE methyltransferase from Saccharop olyspora erythraea methylates a 625 nucleotide transcript of domain V as efficiently as it methylates intact 23 S rRNA. By progressively tru ncating domain V, the motif required for specific recognition by the e nzyme has been localized to a helix and single-stranded region adjacen t to A2058. The smallest RNA transcript that shows methyl-accepting ac tivity is a 27-nucleotide stem-loop, corresponding to the 23 S rRNA se quences 2048 to 2063 and 2610 to 2620 (helix 73), with A2058 situated within the hairpin loop. Methylation of A2058 in the truncated RNAs is optimal in the absence of magnesium, and the efficiency of methylatio n is halved by the presence of 2 to 3 mM magnesium. Magnesium serves t o stabilize a conformation in the truncated RNA that prevents efficien t methylation. This contrasts to the intact domain V RNA,; where 2 mM magnesium ions support a conformation at A2058 that is most readily re cognized by ErmE. Methylation of domain V RNA is generally far less su sceptible to ionic conditions than the truncated RNAs. The effects of monovalent cations on the methylation of truncated transcripts suggest that RNA structures outside helix 73 support the ErmE interaction. Ho wever, interaction with these structures is not essential for specific ErmE recognition of A2058. (C) 1998 Academic Press.