Vester B, Nielsen A K, Hansen L H, Douthwaite S
RNA Regulation Centre Department of Molecular Biology, University of Copenhagen, Denmark.
J Mol Biol. 1998 Sep 18;282(2):255-64. doi: 10.1006/jmbi.1998.2024.
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.
细菌23S rRNA结构域V中腺嘌呤2058（A2058，大肠杆菌编号）的N-6位经Erm甲基转移酶进行二甲基化可赋予对大环内酯-林可酰胺-链阳霉素B（MLS）类抗生素的抗性。来自糖多孢红霉菌的ErmE甲基转移酶对结构域V的625个核苷酸转录本进行甲基化的效率与对完整23S rRNA进行甲基化的效率相同。通过逐步截短结构域V，该酶特异性识别所需的基序已定位到与A2058相邻的一个螺旋和单链区域。表现出甲基接受活性的最小RNA转录本是一个27个核苷酸的茎环，对应于23S rRNA序列2048至2063和2610至2620（螺旋73），A2058位于发夹环内。截短RNA中A2058的甲基化在无镁条件下最为理想，2至3 mM镁的存在会使甲基化效率减半。镁用于稳定截短RNA中的一种构象，从而阻止有效甲基化。这与完整的结构域V RNA形成对比，在完整结构域V RNA中，2 mM镁离子支持A2058处的一种构象，这种构象最容易被ErmE识别。结构域V RNA的甲基化通常比截短的RNA对离子条件的敏感性低得多。单价阳离子对截短转录本甲基化的影响表明，螺旋73之外的RNA结构支持ErmE相互作用。然而，与这些结构的相互作用对于ErmE对A2058的特异性识别并非必不可少。
