Metal interactions with a GAAA RNA tetraloop characterized by (31)P NMR and phosphorothioate substitutions.
Metal interactions with a GAAA RNA tetraloop characterized by (31)P NMR and phosphorothioate substitutions.
复制标题
金属与 GAAA RNA 四环的相互作用通过 (31)P NMR 和硫代磷酸酯取代进行表征。
DOI：
10.1021/bi000140l
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
DeRose,VJ
中科院分区：
文献类型：
--
作者：
Maderia,M;Horton,TE;DeRose,VJ
5 '-GAAA-3'四环中的一个金属位点是一个稳定的、遗传上保守的RNA基序，利用31 P NMR光谱和硫代磷酸酯修饰进行了研究。与以前的报告类似[Legault，P.，和Pardi，A.（1994）J. Magn. Reson.，Ser. B103，82−86]，12个核苷酸的茎环序列5 '-GGCCGAAAGGCC-3'的31 P NMR谱显示出每个磷酸二酯键的分辨特征。用Mg 2+滴定导致这些31 P特征的子集的明显移位，其被分配给磷酸二酯5'至A6、A7和G5。用Co（NH3）63+滴定仅引起A6特征的轻微高场位移，表明由Mg 2+引起的变化是由于内层金属-磷酸盐配位。在四环的三个A残基中的每一个的5'处酶促引入的Rp-硫代磷酸酯取代提供了良好分辨的31 P NMR特征，观察到在Cd 2+而不是Mg 2+存在下发生位移，同样与金属磷酸盐位点一致。使用在环区域中具有单个硫代磷酸酯取代的序列5 '-GGGCGAAAGUCC-3'（分离成RpandSpeastereomers）分析31 P NMR光谱，提供了与磷酸酯5'至A7相互作用的内球相互作用，但引起5'至A6扰动的外球或结构效应的证据。在A7的5'端引入Rp-硫代磷酸酯产生了独特的31 P NMR光谱，与随附论文中报道的热力学研究一致，表明这种取代引起了独特的结构。在这些结果和现有结构信息的基础上，使用约束分子动力学模拟对5 '-GAAA-3'四环中的金属位点进行建模。
A metal site in a 5‘-GAAA-3‘ tetraloop, a stabilizing and phylogenetically conserved RNA motif, is explored using31P NMR spectroscopy and phosphorothioate modifications. Similar to previous reports [Legault, P., and Pardi, A. (1994)J. Magn. Reson., Ser. B103, 82−86], the31P NMR spectrum of a 12-nucleotide stem−loop sequence 5‘-GGCCGAAAGGCC-3‘ exhibits resolved features from each of the phosphodiester linkages. Titration with Mg2+results in distinct shifts of a subset of these31P features, which are assigned to phosphodiesters 5‘ to A6, A7, and G5. Titration with Co(NH3)63+causes only a slight upfield shift in the A6 feature, suggesting that changes caused by Mg2+are due to inner-sphere metal−phosphate coordination.Rp-Phosphorothioate substitutions introduced enzymatically 5‘ to each of the three A residues of the tetraloop provide well-resolved31P NMR features that are observed to shift in the presence of Cd2+but not Mg2+, again consistent with a metal−phosphate site. Analysis of31P NMR spectra using the sequence 5‘-GGGCGAAAGUCC-3‘ with single phosphorothioate substitutions in the loop region, separated intoRpandSpdiastereomers, provides evidence for an inner-sphere interaction with the phosphate 5‘ to A7 but outer-sphere or structural effects that cause perturbations 5‘ to A6. Introduction of anRp-phosphorothioate 5‘ to A7 results in a distinct31P NMR spectrum, consistent with thermodynamic studies reported in the accompanying paper that indicate a unique structure caused by this substitution. On the basis of these results and existing structural information, a metal site in the 5‘-GAAA-3‘ tetraloop is modeled using restrained molecular dynamics simulations.