Dynamics of large elongated RNA by NMR carbon relaxation
Dynamics of large elongated RNA by NMR carbon relaxation
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DOI：
10.1021/ja0757982
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发表时间：
2007-12-26
影响因子：
15
通讯作者：
Al-Hashimi, Hashim M.
中科院分区：
文献类型：
--
作者：
Hansen, Alexandar L.;Al-Hashimi, Hashim M.
我们提出了一种 NMR 策略，用于表征均匀 C-13/N-15 标记的 RNA 中的皮秒至纳秒内部运动，该策略将质子化碱基（C2、C5、C6 和 C8）和糖（C1'）碳的 R-1、R-i rho 和异核 C-13{H-1} NOE 的测量与用于解耦内部的结构域延伸策略相结合。 整体运动和残余偶极耦合 (RDC) 测量，用于确定轴对称旋转扩散主轴的平均 RNA 整体构象和方向。 TROSY 检测的脉冲序列用于精确测量大 RNA 中核碱基碳 R-1 和 R-1 rho 速率。使用无模型形式主义对弛豫数据进行分析，该形式主义考虑了总体旋转扩散的非常高的各向异性（D 比率近似于 4.7）、核碱基 CSA 的不对称性以及 C - C、C - H 偶极和 CSA 相互作用的非共线性，假设给定碳的所有相互作用张量都经历相同的各向同性内部运动。该方法在游离形式和与配体精氨酰胺 (ARG) 结合的延长的 HIV-1 TAR RNA（tau(m) 大约 18 ns）上进行了演示和验证。结果表明，同时ARG结合降低了集体的振幅。结合口袋处的螺旋运动和局部迁移率，它导致连接两个螺旋的“间隔子”凸出残基的局部迁移率急剧增加，这两个螺旋在 ARG 结合状态下经历几乎不受限制的内部运动（S-2 近似于 0.2）。我们的结果建立了定量研究 RNA 动力学的能力，这些 RNA 比通常通过 NMR 碳弛豫研究的更大且更具各向异性。
We present an NMR strategy for characterizing picosecond-to-nanosecond internal motions in uniformly C-13/N-15-labeled RNAs that combines measurements of R-1, R-i rho, and heteronuclear C-13{H-1} NOEs for protonated base (C2, C5, C6, and C8) and sugar (C1') carbons with a domain elongation strategy for decoupling internal from overall motions and residual dipolar coupling (RDC) measurements for determining the average RNA global conformation and orientation of the principal axis of the axially symmetric rotational diffusion. TROSY-detected pulse sequences are presented for the accurate measurement of nucleobase carbon R-1 and R-1 rho rates in large RNAs. The relaxation data is analyzed using a model free formalism which takes into account the very high anisotropy of overall rotational diffusion (D-ratio approximate to 4.7), asymmetry of the nucleobase CSAs and noncollinearity of C - C, C - H dipolar and CSA interactions under the assumption that all interaction tensors for a given carbon experience identical isotropic internal motions. The approach is demonstrated and validated on an elongated HIV-1 TAR RNA (tau(m)approximate to 18 ns) both in free form and bound to the ligand argininamide (ARG). Results show that, while ARG binding reduces the amplitude of collective. helix motions and local mobility at the binding pocket, it leads to a drastic increase in the local mobility of '' spacer '' bulge residues linking the two helices which undergo virtually unrestricted internal motions (S-2 approximate to 0.2) in the ARG bound state. Our results establish the ability to quantitatively study the dynamics of RNAs which are significantly larger and more anisotropic than customarily studied by NMR carbon relaxation.