Departments of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Proc Natl Acad Sci U S A. 2010 Feb 9;107(6):2479-84. doi: 10.1073/pnas.0906319107. Epub 2010 Jan 25.
Functionally critical metals interact with RNA through complex coordination schemes that are currently difficult to visualize at the atomic level under solution conditions. Here, we report a new approach that combines NMR and XAS to resolve and characterize metal binding in the most highly conserved P4 helix of ribonuclease P (RNase P), the ribonucleoprotein that catalyzes the divalent metal ion-dependent maturation of the 5' end of precursor tRNA. Extended X-ray absorption fine structure (EXAFS) spectroscopy reveals that the Zn(2+) bound to a P4 helix mimic is six-coordinate, with an average Zn-O/N bond distance of 2.08 A. The EXAFS data also show intense outer-shell scattering indicating that the zinc ion has inner-shell interactions with one or more RNA ligands. NMR Mn(2+) paramagnetic line broadening experiments reveal strong metal localization at residues corresponding to G378 and G379 in B. subtilis RNase P. A new "metal cocktail" chemical shift perturbation strategy involving titrations with , Zn(2+), and confirm an inner-sphere metal interaction with residues G378 and G379. These studies present a unique picture of how metals coordinate to the putative RNase P active site in solution, and shed light on the environment of an essential metal ion in RNase P. Our experimental approach presents a general method for identifying and characterizing inner-sphere metal ion binding sites in RNA in solution.
功能关键金属通过复杂的配位方案与 RNA 相互作用，目前在溶液条件下很难在原子水平上可视化。在这里，我们报告了一种新的方法，该方法结合了 NMR 和 XAS 来解析和表征核糖核酸酶 P（RNase P）中最高度保守的 P4 螺旋中的金属结合，RNase P 是一种催化前体 tRNA 5'端双价金属离子依赖性成熟的核糖核蛋白。扩展的 X 射线吸收精细结构（EXAFS）光谱表明，与 P4 螺旋模拟物结合的 Zn(2+) 是六配位的，平均 Zn-O/N 键距离为 2.08 A。EXAFS 数据还显示出强烈的外壳散射，表明锌离子与一个或多个 RNA 配体具有内壳相互作用。NMR Mn(2+) 顺磁线宽实验揭示了在枯草芽孢杆菌 RNase P 中对应于 G378 和 G379 的残基处存在强烈的金属定位。一种新的“金属鸡尾酒”化学位移扰动策略涉及与 Zn(2+) 和 Mn(2+) 的滴定，并证实了与残基 G378 和 G379 的内壳金属相互作用。这些研究提供了一个关于金属在溶液中如何与假定的 RNase P 活性位点配位的独特画面，并阐明了 RNase P 中必需金属离子的环境。我们的实验方法为在溶液中鉴定和表征 RNA 中的内壳金属离子结合位点提供了一种通用方法。
