Abstract
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.
Highlights
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 the 3’ end of appropriate tRNAs [1]
A vast majority of tRNAs were complexed at low enzyme concentration (3 and 5 μM), suggesting that yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) interacts with tRNA Arg species and binds readily to noncognate tRNAs
MIST is a non-cellular assay, based on purified molecular partners, it integrates essential in vivo parameters such as tRNA stoichiometry as well as the presence of post-transcriptional modifications
Summary
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 the 3’ end of appropriate tRNAs [1]. AARSs are divided into two classes, of 10 members each, based on structural and sequence features of their active sites [2, 3]. They aminoacylate tRNA in two steps: amino acids are first activated at the expense of ATP followed by the transfer of the amino acid moieties onto tRNAs. Some AARSs such as Arginyl-tRNA synthetases (ArgRS) require the cognate tRNA to synthesize aminoacyl-AMP in the first step [4].
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