title: Isoacceptor specific characterization of tRNA aminoacylation and misacylation in vivo.
abstract: Amino acid misincorporation during protein synthesis occurs due to misacylation of tRNAs or defects in decoding at the ribosome. While misincorporation of amino acids has been observed in a variety of contexts, less work has been done to directly assess the extent to which specific tRNAs are misacylated in vivo, and the identity of the misacylated amino acid moiety. Here we describe tRNA isoacceptor specific aminoacylation profiling (ISAP), a method to identify and quantify the amino acids attached to a tRNA species in vivo. ISAP allows compilation of aminoacylation profiles for specific isoacceptors tRNAs. To demonstrate the efficacy and broad applicability of ISAP, tRNAPheand tRNATyrspecies were isolated from total aminoacyl-tRNA extracted from both yeast and Escherichia coli. Isolated aminoacyl-tRNAs were washed until free of detectable unbound amino acid and subsequently deacylated. Free amino acids from the deacylated fraction were then identified and quantified by mass spectrometry. Using ISAP allowed quantification of the effects of quality control on the accumulation of misacylated tRNA species under different growth conditions.
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA; Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Kyle
authors.initials: K
authors.lastname: Mohler
authors.affiliation: The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Mann
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA; Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA. Electronic address: ibba.1@osu.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Ibba
mesh_terms: D001710:Biotin / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D013058:Mass Spectrometry; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010649:Phenylalanine / Q000302:isolation & purification / Q000378:metabolism*; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000235:genetics / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D019809:Streptavidin / Q000737:chemistry; D046249:Transfer RNA Aminoacylation; D014443:Tyrosine / Q000302:isolation & purification / Q000378:metabolism*; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*
chemical_list: D015342:DNA Probes; D012360:RNA, Transfer, Phe; D012365:RNA, Transfer, Tyr; D014443:Tyrosine; D010649:Phenylalanine; D001710:Biotin; D019809:Streptavidin; D014448:Tyrosine-tRNA Ligase; D010652:Phenylalanine-tRNA Ligase
keywords: Aminoacylation; Isoacceptor; Misacylation; tRNA