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首页> 外文期刊>Journal of Molecular Biology >Analysis of the Cleavage Mechanism by Protein-Only RNase P Using Precursor tRNA Substrates with Modifications at the Cleavage Site
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Analysis of the Cleavage Mechanism by Protein-Only RNase P Using Precursor tRNA Substrates with Modifications at the Cleavage Site

机译：使用前体tRNA底物在切割位点修饰的纯蛋白质RNase P切割机理的分析。

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摘要

Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5'-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein only RNase P (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here, we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana (AtPRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, with either Rp- or Sp-phosphorothioate, or 2'-deoxy, 2'-fluoro, 2'-amino, or 2'-O-methyl substitutions. We observed a small but robust rescue effect of Sp-phosphorothioate-modified pre-tRNA in the presence of thiophilic Cd2+ ions, consistent with metal-ion coordination to the (pro-)Sp-oxygen during catalysis. Sp-phosphorothioate, 2'-deoxy, 2'-amino, and 2'-O-methyl modification redirected the cleavage mainly to the next unmodified phosphodiester in the 5'-direction. Our findings are in line with the 2'-OH substituent at nucleotide - 1 being involved in an H-bonding acceptor function. In contrast to bacterial RNase P, AtPRORP3 was found to be able to utilize the canonical and upstream cleavage site with similar efficiency (corresponding to reduced cleavage fidelity), and the two cleavage pathways appear less interdependent than in the bacterial RNA-based system. (C) 2016 Elsevier Ltd. All rights reserved.
机译：核糖核酸酶P（RNase P）是一种酶，可在生活的所有域中从tRNA转录本中进行核酸内切去除5'前体序列。 RNase P活性是核糖核蛋白（RNP）或仅蛋白质的RNase P（PRORP）酶，这引发了有关这些结构上不同的酶类别用于催化相同类型反应的机制策略的问题。在这里，我们分析了拟南芥PRORP3（AtPRORP3）使用前体tRNA（pre-tRNAs）的动力学和切割位点选择，在标准切割位点进行了单独修饰，Rp-或Sp-硫代磷酸酯或2'-脱氧，2'-氟，2'-氨基或2'-O-甲基取代。我们观察到在亲硫的Cd2 +离子存在下，Sp-硫代磷酸酯修饰的pre-tRNA具有很小但很强的拯救作用，这与催化过程中金属离子对（pro）Sp-氧的配位相一致。 Sp-硫代磷酸酯，2'-脱氧，2'-氨基和2'-O-甲基修饰主要在5'方向上将切割重定向至下一个未修饰的磷酸二酯。我们的发现与核苷酸-1的2'-OH取代基参与H键受体功能相符。与细菌RNase P相比，发现AtPRORP3能够以相似的效率利用标准的和上游的切割位点（对应于降低的切割保真度），并且这两个切割途径的依赖性比基于细菌RNA的系统少。 （C）2016 Elsevier Ltd.保留所有权利。

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