Poster abstracts
Poster number 82 submitted by Rebecca Mann
Isoacceptor specific characterization of tRNA aminoacylation and misacylation in vivo
Rebecca Mann (Department of Microbiology Ohio State University ), Kyle Mohler (Department of Microbiology Ohio State University), Michael Ibba (Department of Microbiology Ohio State University )
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 isoacceptor tRNAs. To demonstrate the efficacy and broad applicability of ISAP, tRNAPhe and tRNATyr species 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.
References:
Reynolds, N.M., Lazazzera, B.A., and Ibba, M. (2010) Cellular mechanisms that control mistranslation. Nature Reviews 2010, 8, 849-856.
Mohler, K., Mann, R. and Ibba, M. (2017) Isoacceptor specific characterization of tRNA aminoacylation and misacylation in vivo. Methods, 113:127-131.
Keywords: tRNA, aaRS