Structure and thermodynamics of metal binding in the P5 helix of a group I intron ribozyme
Structure and thermodynamics of metal binding in the P5 helix of a group I intron ribozyme
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DOI：
10.1006/jmbi.1999.2867
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发表时间：
1999-07-02
影响因子：
5.6
通讯作者：
Tinoco, I
中科院分区：
文献类型：
--
作者：
Colmenarejo, G;Tinoco, I
用核磁共振法测定了RNA发夹的溶液结构，该发夹模拟了I组内含子的P5螺旋，与Co（NH 3）（6）（3+）络合。Co（NH3）（6）（3+）具有与Mg（H2O）（6）（2+）非常接近的几何形状，其用于鉴定和表征RNA中的Mg 2+结合位点。强而正的分子间核Overhauser效应（NOE）交叉峰定义了一种特定的复合物，其中Co（NH3）（6）（3+）分子位于串联G.U碱基对的大沟中。RNA的结构特征是两个G.U碱基对之间的扭转角非常低，提供了一个平坦和狭窄的大沟。Co（NH3）（6）（3+）尽管高度局部化，但可以以多种方式自由旋转成氢键，与尿嘧啶碱基的O 4原子以及鸟嘌呤碱基的N7和O 6形成氢键。序列中其他质子的负和小NOE交叉峰揭示了非特异性或离域相互作用，其特征在于钴离子的高迁移率。P5的Mn 2+滴定显示形成金属离子结合位点的G.U碱基对的质子的特异性增宽，与来自Co（NH3）（6）（3+）的NOE数据一致。通过在用金属离子滴定RNA期间监测亚氨基质子化学位移来确定Co（NH3）（6）（3+）和Mg 2+与P5的相互作用的结合常数。Co（NH3）（6）（3+）的解离常数约为0.1 mM，Mg 2+的解离常数约为1 mM。结合研究进行了相应的串联G.U碱基对的三个方向的序列的突变体。Co（NH3）（6）（3+）和Mg 2+对串联G.U碱基对的亲和力强烈依赖于它们的序列;这种差异可以根据相应金属离子-RNA复合物的不同结构来理解。如预期的，G. C或A.U取代G.U对也影响结合。这些结构和热力学的结果提供了系统的新信息，主要沟金属离子在RNA中的结合。(C)北京：科学出版社.
The solution structure of an RNA hairpin modelling the P5 helix of a group I intron, complexed with Co(NH3)(6)(3+), has been determined by nuclear magnetic resonance. Co(NH3)(6)(3+), which possesses a geometry very close to Mg(H2O)(6)(2+), was used to identify and characterize a Mg2+ binding site in the RNA. Strong and positive intermolecular nuclear Overhauser effect (NOE) cross-peaks define a specific complex in which the Co(NH3)(6)(3+) molecule is in the major groove of tandem G.U basepairs. The structure of the RNA is characterized by a very low twist angle between the two G.U base-pairs, providing a flat and narrowed major groove. The Co(NH3)(6)(3+), although highly localized, is free to rotate to hydrogen bond in several ways to the O4 atoms of the uracil bases and to N7 and O6 of the guanine bases. Negative and small NOE cross-peaks to other protons in the sequence reveal a non-specific or delocalized interaction, characterized by a high mobility of the cobalt ion. Mn2+ titrations of P5 show specific broadening of protons of the G.U basepairs that form the metal ion binding site, in agreement with the NOE data from Co(NH3)(6)(3+). Binding constants for the interaction of Co(NH3)(6)(3+) and of Mg2+ to P5 were determined by monitoring imino proton chemical shifts during titration of the RNA with the metal ions. Dissociation constants are on the order of 0.1 mM for Co(NH3)(6)(3+) and 1 mM for Mg2+. Binding studies were done on mutants with sequences corresponding to the three orientations of tandem G.U base-pairs. The affinities of Co(NH3)(6)(3+) and Mg2+ for the tandem G.U base-pairs depend strongly on their sequences; the differences can be understood in terms of the different structures of the corresponding metal ion-RNA complexes. Substitution of G.C or A.U for G.U pairs also affected the binding, as expected. These structural and thermodynamic results provide systematic new information about major groove metal ion binding in RNA. (C) 1999 Academic Press.