Antibiotic inhibition of RNA catalysis:: Neomycin B binds to the catalytic core of the td group I intron displacing essential metal ions
Antibiotic inhibition of RNA catalysis:: Neomycin B binds to the catalytic core of the td group I intron displacing essential metal ions
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DOI：
10.1006/jmbi.1998.2035
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发表时间：
1998-09-25
影响因子：
5.6
通讯作者：
Schroeder, R
中科院分区：
文献类型：
--
作者：
Hoch, I;Berens, C;Schroeder, R
氨基糖苷类抗生素新霉素B在翻译过程中会导致遗传密码的误读，并抑制几种核酶。T4噬菌体胸苷合成酶(TD)基因产生的I组自剪接内含子就是其中之一。在这里，我们报道了新霉素B是如何与抑制体外剪接的内含子RNA结合的。足迹实验确定了新霉素B的两个主要保护区域：一个在茎P4和P5之间的内环中，另一个在靠近C结合位点的催化核心中。突变分析将后者定义为抑制部位。剪接抑制强烈地依赖于pH和镁离子浓度，这表明静电作用和二价金属离子的竞争。用Fe2+诱导的RNA骨架羟基自由基(Fe-OH)裂解来监测新霉素对金属离子亲和力的影响。新霉素B保护了催化核心中的几个位置不被Fe-OH裂解，这表明在抗生素的存在下金属离子被置换。P7茎中膨胀的核苷酸的突变使剪接对新霉素产生抗性，该位置被新霉素B强烈保护，使其不受Fe-OH切割的影响，并被认为参与了必需金属离子的结合。这些结果使得新霉素能够对接到3D模型中I组内含子的核心。(C)1998年学术出版社。
The aminoglycoside antibiotic neomycin B induces misreading of the genetic code during translation and inhibits several ribozymes. The self-splicing group I intron derived from the T4 phage thymidylate synthase (td) gene is one of these. Here we report how neomycin B binds to the intron RNA inhibiting splicing in vitro. Footprinting experiments identified two major regions of protection by neomycin B: one in the internal loop between the stems P4 and P5 and the other in the catalytic core close to the C-binding site. Mutational analyses defined the latter as the inhibitory site. Splicing inhibition is strongly dependent on pH and Mg2+ concentration, suggesting electrostatic interactions and competition with divalent metal ions. Fe2+-induced hydroxyl radical (Fe-OH) cleavage of the RNA backbone was used to monitor neomycin-mediated changes in the proximity of the metal ions. Neomycin B protected several positions in the catalytic core from Fe-OH cleavage, suggesting that metal ions are displaced in the presence of the antibiotic. Mutation of the bulged nucleotide in the P7 stem, a position which is strongly protected by neomycin B from Fe-OH cleavage and which has been proposed to be involved in binding an essential metal ion, renders splicing resistant to neomycin. These results allowed the docking of neomycin to the core of the group I intron in the 3D model. (C) 1998 Academic Press.