Mn2+−Nitrogen Interactions in RNA Probed by Electron Spin−Echo Envelope Modulation Spectroscopy: Application to the Hammerhead Ribozyme
Mn2+−Nitrogen Interactions in RNA Probed by Electron Spin−Echo Envelope Modulation Spectroscopy: Application to the Hammerhead Ribozyme
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电子自旋回波包络调制光谱探测 RNA 中 Mn2+−氮相互作用：在锤头核酶中的应用
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发表时间：
1999
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通讯作者：
V. DeRose
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作者：
S. Morrissey;T. Horton;C. V. Grant;C. G. Hoogstraten;R. Britt;V. DeRose
我们报告了电子自旋回波包络调制 (ESEEM) 光谱在结构化 RNA 分子中金属配位环境问题中的应用。 ESEEM 已与 15N-鸟苷标记结合使用，以识别锤头核酶和 Mn2+− 鸟苷单磷酸 (GMP) 复合物中 Mn2+ 位点的氮连接。由与 13 核苷酸 DNA 底物链退火的 34 核苷酸 RNA 酶链组成的锤头核酶复合物以 Mn2+ 的 1:1 复合物形式置于 1 M NaCl 中，先前确定的条件是填充单个高亲和力 Mn2+ 位点（Horton, T. E.；Clardy, R. D.；DeRose, V. J. Biochemistry 1998, 51, 18094−18108）。对于自然丰度的 14N 锤头样品，检测到来自几个低频特征的电子自旋回波的显着调制。在 3600 G 时，主要的锤头三脉冲 ESEEM 特征出现在 0.6、1.9、2.5 和 5.2 MHz 处，并且与相同条件下的 Mn2+−GMP 复合物几乎相同。对于一个...
We report application of electron spin−echo envelope modulation (ESEEM) spectroscopy to the problem of metal coordination environments in structured RNA molecules. ESEEM has been used in conjunction with 15N-guanosine labeling to identify nitrogen ligation to a Mn2+ site in a hammerhead ribozyme and in Mn2+−model guanosine monophosphate (GMP) complexes. Hammerhead ribozyme complexes consisting of a 34-nucleotide RNA enzyme strand annealed to a 13-nucleotide DNA substrate strand were poised in 1 M NaCl as a 1:1 complex with Mn2+, conditions previously determined to populate a single high-affinity Mn2+ site (Horton, T. E.; Clardy, R. D.; DeRose, V. J. Biochemistry 1998, 51, 18094−18108). Significant modulation of the electron spin−echo from several low-frequency features is detected for the natural-abundance, 14N-hammerhead samples. At 3600 G, the main hammerhead three-pulse ESEEM features arise at 0.6, 1.9, 2.5, and 5.2 MHz and are nearly identical for a Mn2+−GMP complex under the same conditions. For a ri...