McShane Ariel, Hok Eveline, Tomberlin Jensen, Eriani Gilbert, Geslain Renaud
Laboratory of tRNA biology, Department of Biology, College of Charleston, Charleston, South Carolina, United States of America.
Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084, Strasbourg CEDEX, France.
PLoS One. 2016 Feb 4;11(2):e0148460. doi: 10.1371/journal.pone.0148460. eCollection 2016.
Identity determinants are essential for the accurate recognition of transfer RNAs by aminoacyl-tRNA synthetases. To date, arginine determinants in the yeast Saccharomyces cerevisiae have been identified exclusively in vitro and only on a limited number of tRNA Arginine isoacceptors. In the current study, we favor a full cellular approach and expand the investigation of arginine determinants to all four tRNA Arg isoacceptors. More precisely, this work scrutinizes the relevance of the tRNA nucleotides at position 20, 35 and 36 in the yeast arginylation reaction. We built 21 mutants by site-directed mutagenesis and tested their functionality in YAL5, a previously engineered yeast knockout deficient for the expression of tRNA Arg CCG. Arginylation levels were also monitored using Northern blot. Our data collected in vivo correlate with previous observations. C35 is the prominent arginine determinant followed by G36 or U36 (G/U36). In addition, although there is no major arginine determinant in the D loop, the recognition of tRNA Arg ICG relies to some extent on the nucleotide at position 20. This work refines the existing model for tRNA Arg recognition. Our observations indicate that yeast Arginyl-tRNA synthetase (yArgRS) relies on distinct mechanisms to aminoacylate the four isoacceptors. Finally, according to our refined model, yArgRS is able to accommodate tRNA Arg scaffolds presenting N34, C/G35 and G/A/U36 anticodons while maintaining specificity. We discuss the mechanistic and potential physiological implications of these findings.
身份决定因素对于氨酰 - tRNA合成酶准确识别转运RNA至关重要。迄今为止，酿酒酵母中的精氨酸决定因素仅在体外被鉴定出来，并且仅在有限数量的tRNA精氨酸同工受体上。在当前的研究中，我们倾向于采用全细胞方法，并将精氨酸决定因素的研究扩展到所有四种tRNA Arg同工受体。更确切地说，这项工作仔细研究了酵母精氨酸化反应中第20、35和36位tRNA核苷酸的相关性。我们通过定点诱变构建了21个突变体，并在YAL5中测试了它们的功能，YAL5是一个先前构建的缺乏tRNA Arg CCG表达的酵母敲除株。还使用Northern印迹法监测精氨酸化水平。我们在体内收集的数据与先前的观察结果相关。C35是主要的精氨酸决定因素，其次是G36或U36（G/U）36。此外，虽然D环中没有主要的精氨酸决定因素，但tRNA Arg ICG的识别在一定程度上依赖于第20位的核苷酸。这项工作完善了现有的tRNA Arg识别模型。我们的观察结果表明，酵母精氨酰 - tRNA合成酶（yArgRS）依赖于不同的机制来氨酰化这四种同工受体。最后，根据我们完善的模型，yArgRS能够容纳呈现N34、C/G35和G/A/U36反密码子的tRNA Arg支架，同时保持特异性。我们讨论了这些发现的机制和潜在的生理意义。
