Structure-function relationships in the hammerhead ribozyme probed by base rescue
Structure-function relationships in the hammerhead ribozyme probed by base rescue
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DOI：
10.1017/s1355838298980979
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发表时间：
1998-11-01
期刊：
影响因子：
4.5
通讯作者：
Herschlag, D
中科院分区：
文献类型：
--
作者：
Peracchi, A;Matulic-Adamic, J;Herschlag, D
我们先前表明，在锤头状核酶核心中引入脱碱基核苷酸的有害作用在某些情况下可以通过外源添加烧蚀的碱基来减轻，并且不同碱基拯救催化的相对能力可以用于探测核酶结构的功能方面[Peracchi等人，Proc Nat Acad Sci USA 93：11522]。在这里，我们研究救援在四个额外的位置，3，9，12和13，探测过渡态相互作用，并证明基地救援的优势和劣势作为一种工具的结构-功能研究。结果证实了功能性的作用，以前探测诱变组，提供的证据表明，在基态的X-射线结构中观察到的特定的相互作用保持在过渡态，并建议形成在过渡态的其他相互作用是不存在的基态。此外，结果表明，过渡状态的作用，一些群体没有出现在以前的诱变研究中的重要性，大概是因为基地救援有能力揭示相互作用，在传统的诱变被掩盖的局部结构冗余。通过比较脱碱基和苯基核苷酸取代的效果来补充碱基拯救结果。这些结果共同表明，在基态观察到的9、13和14位碱基的堆叠对于过渡态中其他基团的取向是重要的。这些发现增加了我们对锤头状核酶的结构-功能关系的理解，并有助于描绘可能在活性锤头状结构中相对于基态结构进行重排的位置。最后，相对于腺嘌呤和其他碱基，2-甲基腺嘌呤在位置13处的特别有效的拯救表明，在某些情况下，天然碱基修饰可以通过利用折叠RNA中的疏水相互作用提供额外的稳定性。
We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc Nat Acad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.