Potassium ions modulate a G-quadruplex-ribozyme's activity
Potassium ions modulate a G-quadruplex-ribozyme's activity
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DOI：
10.1261/rna.963908
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发表时间：
2008-06-01
期刊：
影响因子：
4.5
通讯作者：
Perreault, Jean-Pierre
中科院分区：
文献类型：
--
作者：
Beaudoin, Jean-Denis;Perreault, Jean-Pierre
丁型肝炎病毒核酶折叠成紧密堆积的三级结构。然而，与其他核酶不同，它似乎不能遵循替代折叠途径。丁型肝炎病毒核酶的分子工程导致了具有内切核糖核酸酶活性的核酶的开发，所述核酶在G-四链体结构（即，G-石英酶）。后者代表了一类全新的核酶。然后产生这种核酶的突变体，以阐明G-四链体结构的存在所引起的切割活性的调节。在各种单周转条件下进行G-石英酶的动力学表征。发现它仅在钾阳离子的存在下才有活性，钾阳离子在形成G-四链体结构的构建块的四个共面鸟嘌呤的定位中充当抗衡离子。G-quartzyme表现为变构核酶，其中钾阳离子作为希尔系数为2.9 +/- 0.2的正效应物。由钾离子的存在引起的构象转变由非活性（关闭）和活性（打开）结构的酶和化学探测支持。这项研究表明，有可能通过添加一种不寻常的稳定结构来干扰丁型肝炎病毒核酶的紧密结构。据我们所知，G-石英酶是唯一的核酶，表现出一个单价阳离子依赖性的活动。
Hepatitis delta virus ribozyme folds into a tightly packed tertiary structure. However, unlike other ribozymes, it does not appear to be able to follow alternative folding pathways. Molecular engineering of the hepatitis delta virus ribozyme led to the development of a ribozyme possessing an endoribonuclease activity that is under the control of a G-quadruplex structure (i.e., a G-quartzyme). This latter species represents an entirely new class of ribozyme. Mutants of this ribozyme were then generated in order to shed light on the modulation of the cleavage activity caused by the presence of the G-quadruplex structure. Kinetic characterization of the G-quartzyme was performed under various single turnover conditions. It was found to be active only in the presence of potassium cations that act as counter ions in the positioning of the four coplanar guanines that form the building block of the G-quadruplex structure. The G-quartzyme behaves as an allosteric ribozyme, with the potassium cations acting as positive effectors with a Hill coefficient of 2.9 +/- 0.2. The conformation transition caused by the presence of the potassium ions is supported by enzymatic and chemical probing of both the inactive (off) and active (on) structures. This study shows that it is possible to interfere with the tight structure of the hepatitis delta virus ribozyme by adding an unusual, stable structure. To our knowledge, the G-quartzyme is the sole ribozyme that exhibits a monovalent cation-dependent activity.