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 Saccharopolyspora erythraea methylates a 625 nucleotide transcript of domain V as efficiently as it methylates intact 23 S rRNA. By progressively truncating domain V, the motif required for specific recognition by the enzyme has been localized to a helix and single-stranded region adjacent to A2058. The smallest RNA transcript that shows methyl-accepting activity is a 27-nucleotide stem-loop, corresponding to the 23 S rRNA sequences 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 methylation is halved by the presence of 2 to 3 mM magnesium. Magnesium serves to stabilize a conformation in the truncated RNA that prevents efficient methylation. This contrasts to the intact domain V RNA, where 2 mM magnesium ions support a conformation at A2058 that is most readily recognized by ErmE. Methylation of domain V RNA is generally far less susceptible 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. However, interaction with these structures is not essential for specific ErmE recognition of A2058.
Vester
B
B
RNA Regulation Centre Department of Molecular Biology, University of Copenhagen, Denmark.
Nielsen
A K
AK
Hansen
L H
LH
Douthwaite
S
S
D000643:Ammonium Chloride / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008745:Methylation / Q000187:drug effects; D008780:Methyltransferases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D000643:Ammonium Chloride; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase; D008274:Magnesium