Tinsley Rebecca A, Harris Dinari A, Walter Nils G
Department of Chemistry, The University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, USA.
Biochemistry. 2004 Jul 20;43(28):8935-45. doi: 10.1021/bi049471e.
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 Mg(2+) 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 Mg(2+) 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 Mg(2+) concentration, essentially amplifying the structural differences observed in the crystal structures. In addition, the precursor has an approximately 2-fold and approximately 13-fold lower Mg(2+) 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 Mg(2+) affinity for both their precursor and product forms. Significantly, the Mg(2+) 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.
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，使得在核酶功能中区分其因果关系变得困难。例如，最近解析的顺式切割基因组丁型肝炎病毒（HDV）核酶前体和产物形式的晶体结构显示，活性位点结合有一个二价金属离子，在催化过程中由于RNA构象变化而释放。这种构象转换与活性位点裂隙中参与催化的碱基C75的重新定位相关，从而控制催化作用。这些发现证实了先前关于HDV核酶反式作用形式的荧光共振能量转移（FRET）数据，该数据发现催化作用伴随着全局构象变化。在这里，我们通过测量反式作用HDV核酶全局构象变化的Mg(2+)依赖性，进一步测试构象转换模型，分别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。我们观察到，在存在和不存在300 mM Na(+)背景的情况下，前体和产物形式在结构和Mg(2+)亲和力方面都存在显著差异。随着Mg(2+)浓度的增加，前体缩短而产物延长，基本上放大了晶体结构中观察到的结构差异。此外，在前体和产物形式的二级和三级结构形成中，前体的Mg(2+)亲和力分别比产物低约2倍和约13倍。我们还比较了C75野生型和催化无活性的C75U突变体，发现它们的前体和产物形式在全局结构和Mg(2+)亲和力方面存在显著差异。值得注意的是，C75野生型的Mg(2+)亲和力比C75U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至关重要的观点一致。因此，出现了一个一致的图景，即二价金属离子和RNA功能基团在影响HDV核酶的结构动力学和催化作用方面紧密交织。
