Magnesium dependence of the amplified conformational switch in the trans-acting hepatitis delta virus ribozyme.
Magnesium dependence of the amplified conformational switch in the trans-acting hepatitis delta virus ribozyme.
复制标题
反式作用丁型肝炎病毒核酶中放大构象开关的镁依赖性。
DOI：
10.1021/bi049471e
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Walter,NilsG
中科院分区：
文献类型：
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作者：
Tinsley,RebeccaA;Harris,DinariA;Walter,NilsG
二价金属离子参与RNA酶（核酶）的结构形成和催化化学的能力使得难以分离它们在核酶功能中的因果关系。例如，最近解析的顺式切割基因组丁型肝炎病毒（HDV）核酶的前体和产物形式的晶体结构显示出结合在活性位点中的二价金属离子，其在催化时由于RNA构象变化而释放。这种构象转换与活性位点裂缝中催化参与的碱基C75的重新定位相关，从而控制催化。这些发现证实了以前的数据，从荧光共振能量转移（FRET）的反式作用形式的HDV核酶，发现一个全球性的构象变化，伴随催化。在这里，我们进一步测试的构象开关模型，通过测量Mg 2+依赖的反式作用HDV核酶的全局构象变化，使用圆二色性和时间分辨FRET作为二级和三级结构形成的互补探针，分别。我们观察到显着差异的结构和Mg 2+亲和力的前体和产品形式，在存在和不存在的300 mM Na+背景。前体缩短，而产品随着Mg 2+浓度的增加而延伸，基本上放大了在晶体结构中观察到的结构差异。此外，在二级和三级结构形成中，前体的Mg 2+亲和力分别比产物低1.2倍和1.13倍。我们还比较了C75野生型与无催化活性的C75 U突变体，发现它们的前体和产物形式在全局结构和Mg 2+亲和力方面存在显着差异。值得注意的是，C75野生型的Mg 2+亲和力比C75 U突变体低1.7 - 2.1倍，这与C75对于导致局部二价金属离子亲和力降低和催化金属释放的催化构象变化是必需的这一观点雅阁。因此，一个一致的画面出现，其中二价金属离子和RNA官能团密切交织在一起，影响HDV核酶的结构动力学和催化作用。
The ability of divalent metal ions to participate in both structure formation and catalytic chemistry of RNA enzymes (ribozymes) has made it difficult to separate their cause and effect in ribozyme function. For example, the recently solved crystal structures of precursor and product forms of the cis-cleaving genomic hepatitis delta virus (HDV) ribozyme show a divalent metal ion bound in the active site that is released upon catalysis due to an RNA conformational change. This conformational switch is associated with a repositioning of the catalytically involved base C75 in the active-site cleft, thus controlling catalysis. These findings confirm previous data from fluorescence resonance energy transfer (FRET) on a trans-acting form of the HDV ribozyme that found a global conformational change to accompany catalysis. Here, we further test the conformational switch model by measuring the Mg2+dependence of the global conformational change of the trans-acting HDV ribozyme, using circular dichroism and time-resolved FRET as complementary probes of secondary and tertiary structure formation, respectively. We observe significant differences in both structure and Mg2+affinity of the precursor and product forms, in the presence and absence of 300 mM Na+background. The precursor shortens while the product extends with increasing Mg2+concentration, essentially amplifying the structural differences observed in the crystal structures. In addition, the precursor has an ∼2-fold and ∼13-fold lower Mg2+affinity than the product in secondary and tertiary structure formation, respectively. We also have compared the C75 wild-type with the catalytically inactive C75U mutant and find significant differences in global structure and Mg2+affinity for both their precursor and product forms. Significantly, the Mg2+affinity of the C75 wild-type is 1.7−2.1-fold lower than that of the C75U mutant, in accord with the notion that C75 is essential for a catalytic conformational change that leads to a decrease in the local divalent metal ion affinity and release of a catalytic metal. Thus, a consistent picture emerges in which divalent metal ions and RNA functional groups are intimately intertwined in affecting structural dynamics and catalysis in the HDV ribozyme.