Eriani Gilbert, Karam Joseph, Jacinto Jomel, Morris Richard Erin, Geslain Renaud
Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, Institut de Biologie Moléculaire et Cellulaire, 67084, Strasbourg, CEDEX, France.
Laboratory of tRNA Biology, Department of Biology, College of Charleston, Charleston, South Carolina, United States of America.
PLoS One. 2015 Jun 11;10(6):e0130042. doi: 10.1371/journal.pone.0130042. eCollection 2015.
Aminoacyl-tRNA synthetases (AARSs) constitute a family of RNA-binding proteins, that participate in the translation of the genetic code, by covalently linking amino acids to appropriate tRNAs. Due to their fundamental importance for cell life, AARSs are likely to be one of the most ancient families of enzymes and have therefore been characterized extensively. Paradoxically, little is known about their capacity to discriminate tRNAs mainly because of the practical challenges that represent precise and systematic tRNA identification. This work describes a new technical and conceptual approach named MIST (Microarray Identification of Shifted tRNAs) designed to study the formation of tRNA/AARS complexes independently from the aminoacylation reaction. MIST combines electrophoretic mobility shift assays with microarray analyses. Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition. In this study we focus on yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) and investigate in depth its ability to discriminate cellular tRNAs. We report that yArgRS in submicromolar concentrations binds cognate and non-cognate tRNAs with a wide range of apparent affinities. In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation. Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.
氨酰-tRNA合成酶（AARSs）是一类RNA结合蛋白家族，它们通过将氨基酸共价连接到合适的tRNA上参与遗传密码的翻译。由于其对细胞生命的根本重要性，AARSs可能是最古老的酶家族之一，因此已得到广泛研究。矛盾的是，人们对它们区分tRNA的能力知之甚少，主要是因为精确和系统地鉴定tRNA存在实际挑战。这项工作描述了一种名为MIST（移位tRNA的微阵列鉴定）的新技术和概念方法，旨在独立于氨酰化反应研究tRNA/AARS复合物的形成。MIST将电泳迁移率变动分析与微阵列分析相结合。尽管MIST是一种非细胞分析方法，但它完全整合了tRNA竞争的概念。在本研究中，我们聚焦于酵母细胞质精氨酰-tRNA合成酶（yArgRS），并深入研究其区分细胞内tRNA的能力。我们报告称，亚微摩尔浓度的yArgRS以广泛的表观亲和力结合同源和非同源tRNA。特别是，我们证明yArgRS优先结合II型tRNA，但不支持它们的错误氨酰化。我们的结果揭示了tRNA/AARS复合物形成的重要新趋势以及潜在的深刻生理意义。
