Gonzalez R L, Tinoco I
Berkeley and Structural Biology Department, Physical Biosciences Division, University of California, Berkeley, CA, 94720-1460, USA.
J Mol Biol. 1999 Jun 25;289(5):1267-82. doi: 10.1006/jmbi.1999.2841.
Identification and characterization of a metal ion binding site in an RNA pseudoknot was accomplished using cobalt (III) hexammine, Co(NH3)63+, as a probe for magnesium (II) hexahydrate, Mg(H2O)62+, in nuclear magnetic resonance (NMR) structural studies. The pseudoknot causes efficient -1 ribosomal frameshifting in mouse mammary tumor virus. Divalent metal ions, such as Mg2+, are critical for RNA structure and function; Mg2+preferentially stabilizes the pseudoknot relative to its constituent hairpins. The use of Co(NH3)63+as a substitute for Mg2+was investigated by ultraviolet absorbance melting curves, NMR titrations of the imino protons, and analysis of NMR spectra in the presence of Mg2+or Co (NH3)63+. The structure of the pseudoknot-Co(NH3)63+complex reveals an ion-binding pocket formed by a short, two-nucleotide loop and the major groove of a stem. Co(NH3)63+stabilizes the sharp loop-to-stem turn and reduces the electrostatic repulsion of the phosphates in three proximal strands. Hydrogen bonds are identified between the Co(NH3)63+protons and non-bridging phosphate oxygen atoms, 2' hydroxyl groups, and nitrogen and oxygen acceptors on the bases. The binding site is significantly different from that previously characterized in the major groove surface of tandem G.U base-pairs, but is similar to those observed in crystal structures of a fragment of the 5 S rRNA and the P5c helix of the Tetrahymena thermophila group I intron. Changes in chemical shifts occurred at the same pseudoknot protons on addition of Mg2+as on addition of Co(NH3)63+, indicating that both ions bind at the same site. Ion binding dissociation constants of approximately 0.6 mM and 5 mM (in 200 mM Na+and a temperature of 15 degrees C) were obtained for Co(NH3)63+and Mg2+, respectively, from the change in chemical shift as a function of metal ion concentration. An extensive array of non-sequence-specific hydrogen bond acceptors coupled with conserved structural elements within the binding pocket suggest a general mode of divalent metal ion stabilization of this type of frameshifter pseudoknot. These results provide new thermodynamic and structural insights into the role divalent metal ions play in stabilizing RNA tertiary structural motifs such as pseudoknots.
在核磁共振（NMR）结构研究中，利用六氨合钴（III）[Co(NH₃)₆³⁺]作为六水合镁（II）[Mg(H₂O)₆²⁺]的探针，完成了对RNA假结中金属离子结合位点的鉴定和表征。该假结在小鼠乳腺肿瘤病毒中导致有效的-1核糖体移码。二价金属离子，如Mg²⁺，对RNA的结构和功能至关重要；相对于其组成发夹结构，Mg²⁺优先稳定假结。通过紫外吸收熔解曲线、亚氨基质子的NMR滴定以及在Mg²⁺或Co(NH₃)₆³⁺存在下的NMR光谱分析，研究了用Co(NH₃)₆³⁺替代Mg²⁺的情况。假结-Co(NH₃)₆³⁺复合物的结构揭示了一个由短的两核苷酸环和一个茎的大沟形成的离子结合口袋。Co(NH₃)₆³⁺稳定了尖锐的环到茎的转折，并减少了三个近端链中磷酸根的静电排斥。在Co(NH₃)₆³⁺质子与非桥连磷酸根氧原子、2'-羟基以及碱基上的氮和氧受体之间发现了氢键。该结合位点与先前在串联G·U碱基对大沟表面所表征的位点有显著不同，但与在5S rRNA片段和嗜热四膜虫I组内含子的P5c螺旋晶体结构中观察到的位点相似。加入Mg²⁺和加入Co(NH₃)₆³⁺时，相同的假结质子发生了化学位移变化，表明两种离子都结合在同一位点。根据化学位移随金属离子浓度的变化，分别得到Co(NH₃)₆³⁺和Mg²⁺的离子结合解离常数约为0.6 mM和5 mM（在200 mM Na⁺和15℃温度下）。结合口袋内大量非序列特异性氢键受体与保守结构元件的结合，提示了二价金属离子稳定这种类型移码假结的一般模式。这些结果为二价金属离子在稳定RNA三级结构基序（如假结）中所起的作用提供了新的热力学和结构见解。
