Recognition Elements in rRNA for the Tylosin Resistance Methyltransferase RlmAII

The methyltransferase RlmAII (formerly TlrB) is found in many Gram-positive bacteria, and methylates the N-1 position of nucleotide G748 within the loop of hairpin 35 in 23S rRNA. Methylation of the rRNA by RlmAII confers resistance to tylosin and other mycinosylated 16-membered ring macrolide antibiotics. We have previously solved the solution structure of hairpin 35 in the conformation that is recognized by the RlmAII methyltransferase from Streptococcus pneumoniae. It was shown that while essential recognition elements are located in hairpin 35, the interactions between RlmAII and hairpin 35 are insufficient on their own to support the methylation reaction. Here we use biochemical techniques in conjunction with heteronuclear/homonuclear nuclear magnetic resonance spectroscopy to define the RNA structures that are required for efficient methylation by RlmAII. Progressive truncation of the rRNA substrate indicated that multiple contacts occur between RlmAII and nucleotides in stem–loops 33, 34 and 35. RlmAII appears to recognize its rRNA target through specific surface shape complementarity at the junction formed by these three helices. This means of recognition is highly similar to that of the orthologous Gram-negative methyltransferase, RlmAI (formerly RrmA), which also interacts with hairpin 35, but methylates at the adjacent nucleotide G745.