NDLI: A Label-Free Assay for Aminoacylation of tRNA

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A Label-Free Assay for Aminoacylation of tRNA

Content Provider	MDPI
Author	Gamper, Howard Hou, Ya-Ming
Copyright Year	2020
Description	Aminoacylation of tRNA generates an aminoacyl-tRNA (aa-tRNA) that is active for protein synthesis on the ribosome. Quantification of aminoacylation of tRNA is critical to understand the mechanism of specificity and the flux of the aa-tRNA into the protein synthesis machinery, which determines the rate of cell growth. Traditional assays for the quantification of tRNA aminoacylation involve radioactivity, either with a radioactive amino acid or with a [3′-32P]-labeled tRNA. We describe here a label-free assay that monitors aminoacylation by biotinylation-streptavidin (SA) conjugation to the α-amine or the α-imine of the aminoacyl group on the aa-tRNA. The conjugated aa-tRNA product is readily separated from the unreacted tRNA by a denaturing polyacrylamide gel, allowing for quantitative measurement of aminoacylation. This label-free assay is applicable to a wide range of amino acids and tRNA sequences and to both classes of aminoacylation. It is more sensitive and robust than the assay with a radioactive amino acid and has the potential to explore a wider range of tRNA than the assay with a [3′-32P]-labeled tRNA. This label-free assay reports kinetic parameters of aminoacylation quantitatively similar to those reported by using a radioactive amino acid, suggesting its broad applicability to research relevant to human health and disease.
Starting Page	1173
e-ISSN	20734425
DOI	10.3390/genes11101173
Journal	Genes
Issue Number	10
Volume Number	11
Language	English
Publisher	MDPI
Publisher Date	2020-10-07
Access Restriction	Open
Subject Keyword	Genes Analytical Chemistry Aminoacyl-trna Synthetases Prolyl-trna Tyrosyl-trna Biotinylation To Aminoacyl-trna Acp3u47 in Trna
Content Type	Text
Resource Type	Article

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