title: Determining the fidelity of tRNA aminoacylation via microarrays.
abstract: The fidelity of tRNA aminoacylation is a critical determinant for the ultimate accuracy of protein synthesis. Although aminoacyl-tRNA synthetases are assumed to consistently maintain high tRNA charging fidelity, recent evidence has demonstrated that the fidelity of the aminoacylation reaction can be actively regulated and liable to change. Accordingly, the ability to conveniently assay the fidelity of tRNA charging is becoming increasingly relevant for studying mistranslation. Here we describe a combined radioactivity and microarray based method that can quantitatively elucidate which individual cognate or noncognate tRNA isoacceptors are charged with amino acid. In this technique, in vitro tRNA charging reactions or in vivo pulse-labeling is performed using a radiolabeled amino acid and tRNA microarrays are used to distinguish tRNA isoacceptors in total tRNA. During the tRNA array hybridization, each tRNA will hybridize to its unique probe and subsequent phosphorimaging of the array can determine which tRNAs were aminoacylated with the radiolabeled amino acid. The method can be used to assess the fidelity of tRNA charging in vivo or in vitro and can be applied to any organism with annotated tRNA genes.
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Chicago, Chicago, IL 60637, USA; Committee on Microbiology, University of Chicago, Chicago, IL 60637, USA.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Schwartz
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Chicago, Chicago, IL 60637, USA; Committee on Microbiology, University of Chicago, Chicago, IL 60637, USA. Electronic address: taopan@uchicago.edu.
authors.forename: Tao
authors.initials: T
authors.lastname: Pan
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D002250:Carbon Radioisotopes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D046228:Microarray Analysis / Q000295:instrumentation*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D011327:Printing / Q000379:methods; D015347:RNA Probes / Q000138:chemical synthesis; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D013462:Sulfur Radioisotopes; D046249:Transfer RNA Aminoacylation; D014316:Tritium
chemical_list: D000596:Amino Acids; D002250:Carbon Radioisotopes; D015347:RNA Probes; D012345:RNA, Transfer, Amino Acid-Specific; D013462:Sulfur Radioisotopes; D014316:Tritium; D000604:Amino Acyl-tRNA Synthetases
keywords: Aminoacylation; Microarray; Mistranslation; tRNA