Conservation of helical structure contributes to functional metal ion interactions in the catalytic domain of ribonuclease P RNA
Conservation of helical structure contributes to functional metal ion interactions in the catalytic domain of ribonuclease P RNA
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DOI：
10.1016/s0022-2836(02)01094-x
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发表时间：
2002-11-29
影响因子：
5.6
通讯作者：
Harris, ME
中科院分区：
文献类型：
--
作者：
Kaye, NM;Zahler, NH;Harris, ME
像蛋白酶一样，催化RNA包含对功能重要的保守结构基序。核糖核酸酶P RNA的催化结构域的一个普遍特征是P1-P4螺旋连接内的凸出螺旋基序。在这里，我们表明，在螺旋P4内的膨胀的核苷酸身份和位置的变化影响催化和底物结合，而一个子集的突变只导致催化缺陷。我们发现，接近凸起的P4中的金属离子配位的网站是重要的催化;移动凸起远端这些网站和删除它有类似的大的影响，而移动它接近这些网站只有一个温和的催化作用。为了测试突变的影响是否与金属离子相互作用有关，我们使用磷酸骨架的铽依赖性切割来探测野生型和突变体核酶中的金属离子结合位点。我们检测裂解在催化结构域内的特定位点，包括螺旋P4和J3/4，这已被证明直接参与金属离子相互作用。引入P4的突变导致铽切割模式的局部变化，这是由于与螺旋交替的金属离子结合构型。此外，凸出缺失突变导致饱和镁水平下的单转换切割速率常数降低100倍，并且对催化重要的镁离子亲和力降低。鉴于凸起缺失引起的P4中的交替铽切割模式，这种利用镁离子进行催化的能力降低似乎是由于核酶催化核心中的局部结构变化，其削弱了P4和J3/4中的金属离子相互作用。因此，这里报道的信息提供了证据，证明P4结构的普遍保守性部分基于对催化重要的金属离子相互作用的优化。(C)2002爱思唯尔科技有限公司版权所有。
Like protein enzymes, catalytic RNAs contain conserved structure motifs important for function. A universal feature of the catalytic domain of ribonuclease P RNA is a bulged-helix motif within the P1-P4 helix junction. Here, we show that changes in bulged nucleotide identity and position within helix P4 affect both catalysis and substrate binding, while a subset of the mutations resulted only in catalytic defects. We find that the proximity of the bulge to sites of metal ion coordination in P4 is important for catalysis; moving the bulge distal to these sites and deleting it had similarly large effects, while moving it proximal to these sites had only a moderate effect on catalysis. To test whether the effects of the mutations are linked to metal ion interactions, we used terbium-dependent cleavage of the phosphate backbone to probe metal ion-binding sites in the wild-type and mutant ribozymes. We detect cleavages at specific sites within the catalytic domain, including helix P4 and J3/4, which have previously been shown to participate directly in metal ion interactions. Mutations introduced into P4 cause local changes in the terbium cleavage pattern due to alternate metal ion-binding configurations with the helix. In addition, a bulge deletion mutation results in a 100-fold decrease in the single turnover cleavage rate constant at saturating magnesium levels, and a reduced affinity for magnesium ions important for catalysis. In light of the alternate terbium cleavage pattern in P4 caused by bulge deletion, this decreased ability to utilize magnesium ions for catalysis appears to be due to localized structural changes in the ribozyme's catalytic core that weaken metal ion interactions in P4 and J3/4. The information reported here, therefore, provides evidence that the universal conservation of the P4 structure is based in part on optimization of metal ion interactions important for catalysis. (C) 2002 Elsevier Science Ltd. All rights reserved.