title: MIST, a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases.
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.
authors.affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, Institut de Biologie Moléculaire et Cellulaire, 67084, Strasbourg, CEDEX, France.
authors.forename: Gilbert
authors.initials: G
authors.lastname: Eriani
authors.affiliation: Laboratory of tRNA Biology, Department of Biology, College of Charleston, Charleston, South Carolina, United States of America.
authors.forename: Joseph
authors.initials: J
authors.lastname: Karam
authors.affiliation: Laboratory of tRNA Biology, Department of Biology, College of Charleston, Charleston, South Carolina, United States of America.
authors.forename: Jomel
authors.initials: J
authors.lastname: Jacinto
authors.affiliation: Laboratory of tRNA Biology, Department of Biology, College of Charleston, Charleston, South Carolina, United States of America.
authors.forename: Erin
authors.initials: E
authors.lastname: Morris Richard
authors.affiliation: Laboratory of tRNA Biology, Department of Biology, College of Charleston, Charleston, South Carolina, United States of America.
authors.forename: Renaud
authors.initials: R
authors.lastname: Geslain
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D049148:Aminoacylation; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D046228:Microarray Analysis / Q000379:methods*; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases