Exploring purine N7 interactions via atomic mutagenesis: The group I ribozyme as a case study
Exploring purine N7 interactions via atomic mutagenesis: The group I ribozyme as a case study
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DOI：
10.1261/rna.031567.111
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发表时间：
2012-06-01
期刊：
影响因子：
4.5
通讯作者：
Herschlag, Daniel
中科院分区：
文献类型：
--
作者：
Forconi, Marcello;Benz-Moy, Tara;Herschlag, Daniel
原子诱变已成为揭示复杂RNA分子中特定相互作用的有力工具。Bass和Cech对I组内含子自剪接中外源鸟苷亲核试剂类似物的早期广泛研究表明，结构-功能关系类似于蛋白质配体，并提供了由RNA组成的结构良好的底物结合位点的有力证据。随后的功能和结构研究证实了这些相互作用的位点，并扩展了我们对它们的理解，但有一个值得注意的例外。尽管在最初的研究中，7-甲基鸟苷并不影响反应性，但随后的一项研究揭示了看似更保守的7-二氮杂取代的有害影响。在这里，我们研究这个悖论，研究这些和其他类似物与更彻底表征核酶衍生自四膜虫组I内含子。我们发现，相对于同源的外源鸟苷亲核试剂，7-去氮杂取代降低了20倍的结合，而与7-甲基和8-氮杂-7-去氮杂取代的结合和反应没有影响。这些和其他结果表明，外源鸟苷的N7原子与核酶之间没有功能上重要的接触。相反，它们与N7取代引入的间接影响是一致的，这些间接影响是对结合重要的堆叠相互作用和/或溶剂化。本文使用的一组类似物在破译核酸相互作用以及它们如何通过其他RNA和RNA/蛋白质复合物的反应周期变化方面应该是有价值的。
Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure-function relationships analogous to those seen for protein ligands and provided strong evidence for a well-formed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by similar to 20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes.