Vester B, Douthwaite S
Department of Molecular Biology, Odense University, Denmark.
J Bacteriol. 1994 Nov;176(22):6999-7004. doi: 10.1128/jb.176.22.6999-7004.1994.
The ErmE methyltransferase from the erythromycin-producing actinomycete Saccharopolyspora erythraea dimethylates the N-6 position of adenine 2058 in domain V of 23S rRNA. This modification confers resistance to erythromycin and to other macrolide, lincosamide, and streptogramin B antibiotics. We investigated what structural elements in 23S rRNA are required for specific recognition by the ErmE methyltransferase. The ermE gene was cloned into R1 plasmid derivatives, providing a means of inducible expression in Escherichia coli. Expression of the methyltransferase in vivo confers resistance to erythromycin and clindamycin. The degree of resistance corresponds to the level of ermE expression. In turn, ermE expression also correlates with the proportion of 23S rRNA molecules that are dimethylated at adenine 2058. The methyltransferase was isolated in an active, concentrated form from E. coli, and the enzyme efficiently modifies 23S rRNA in vitro. Removal of most of the 23S rRNA structure, so that only domain V (nucleotides 2000 to 2624) remains, does not affect the efficiency of modification by the methyltransferase. In addition, modification still occurs after the rRNA tertiary structure has been disrupted by removal of magnesium ions. We conclude that the main features that are specifically recognized by the ErmE methyltransferase are displayed within the primary and secondary structures of 23S rRNA domain V.
来自产红霉素的放线菌糖多孢红霉菌的ErmE甲基转移酶可使23S rRNA结构域V中腺嘌呤2058的N-6位发生二甲基化。这种修饰赋予了对红霉素以及其他大环内酯类、林可酰胺类和链阳霉素B抗生素的抗性。我们研究了23S rRNA中的哪些结构元件是ErmE甲基转移酶进行特异性识别所必需的。将ermE基因克隆到R1质粒衍生物中，提供了一种在大肠杆菌中诱导表达的方法。甲基转移酶在体内的表达赋予了对红霉素和克林霉素的抗性。抗性程度与ermE的表达水平相对应。反过来，ermE的表达也与在腺嘌呤2058处发生二甲基化的23S rRNA分子的比例相关。从大肠杆菌中以活性浓缩形式分离出甲基转移酶，该酶在体外能有效地修饰23S rRNA。去除大部分23S rRNA结构，使得仅保留结构域V（核苷酸2000至2624），并不影响甲基转移酶的修饰效率。此外，在通过去除镁离子破坏rRNA三级结构后，修饰仍会发生。我们得出结论，ErmE甲基转移酶进行特异性识别的主要特征表现在23S rRNA结构域V的一级和二级结构中。
