Structural characterization of the 69 nucleotide potato spindle tuber viroid left-terminal domain by NMR and thermodynamic analysis
Structural characterization of the 69 nucleotide potato spindle tuber viroid left-terminal domain by NMR and thermodynamic analysis
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DOI：
10.1016/j.jmb.2003.10.015
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发表时间：
2003-12-05
影响因子：
5.6
通讯作者：
Grzesiek, S
中科院分区：
文献类型：
--
作者：
Dingley, AJ;Steger, G;Grzesiek, S
马铃薯纺锤体块茎 RNA 类病毒 (PSTVd) 的 69 个核苷酸左端结构域 (TO) 构成其五个结构元件之一。由于双重互补序列重复，为 T 二级结构提出了两种可能的构象：拉长杆和分叉形式。在本研究中，设计了两个 TL 突变体，消除了序列重复的对称性，并确保分叉或拉长杆构象在热力学上是热力学的。基于 H-1-H-1 NOESY 和异核 H-1-N-15 HSQC 高分辨率 NMR 光谱，亚氨基 H-1 和 N-15 共振被指定为天然 TL 结构域的细长杆形式，温度梯度凝胶电泳和 UV 熔化实验证实了这些结果。对其他 Pospiviroidae 家族成员的 TL 结构域的结构和序列分析表明，绝大多数类病毒在热力学上倾向于细长杆状结构 (C) 2003 Elsevier Ltd。保留所有权利。
The 69 nucleotide left-terminal domain (TO of the potato spindle tuber RNA viroid (PSTVd) constitutes one of its five structural elements. Due to a twofold complementary sequence repeat, two possible conformations are proposed for the T, secondary structure; an elongated-rod and a bifurcated form. In the present study, two TL mutants were designed that remove the symmetry of the sequence repeats and ensure that either the bifurcated or the elongated-rod conformation is thermodynamically favored. Imino H-1 and N-15 resonances were assigned for both mutants and the native TL domain based on H-1-H-1 NOESY and heteronuclear H-1-N-15 HSQC high-resolution NMR spectra. The NMR secondary structure analysis of all constructs establishes unambiguously the elongated-rod form as the secondary structure of the native TL domain. Temperature-gradient gel electrophoresis and UV melting experiments corroborate these results. A combined secondary structure and sequence analysis of TL domains of other Pospiviroidae family members indicates that the elongated-rod form is thermodynamically favored for the vast majority of these viroids. (C) 2003 Elsevier Ltd. All rights reserved.