A Label-Free Assay for Aminoacylation of tRNA
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 ra...
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doaj-881b202719be42408cf13570833194622020-11-25T03:35:33ZengMDPI AGGenes2073-44252020-10-01111173117310.3390/genes11101173A Label-Free Assay for Aminoacylation of tRNAHoward Gamper0Ya-Ming Hou1Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USADepartment of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USAAminoacylation 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′-<sup>32</sup>P]-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′-<sup>32</sup>P]-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.https://www.mdpi.com/2073-4425/11/10/1173aminoacyl-tRNA synthetasesprolyl-tRNAtyrosyl-tRNAbiotinylation to aminoacyl-tRNAacp<sup>3</sup>U47 in tRNA |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Howard Gamper Ya-Ming Hou |
spellingShingle |
Howard Gamper Ya-Ming Hou A Label-Free Assay for Aminoacylation of tRNA Genes aminoacyl-tRNA synthetases prolyl-tRNA tyrosyl-tRNA biotinylation to aminoacyl-tRNA acp<sup>3</sup>U47 in tRNA |
author_facet |
Howard Gamper Ya-Ming Hou |
author_sort |
Howard Gamper |
title |
A Label-Free Assay for Aminoacylation of tRNA |
title_short |
A Label-Free Assay for Aminoacylation of tRNA |
title_full |
A Label-Free Assay for Aminoacylation of tRNA |
title_fullStr |
A Label-Free Assay for Aminoacylation of tRNA |
title_full_unstemmed |
A Label-Free Assay for Aminoacylation of tRNA |
title_sort |
label-free assay for aminoacylation of trna |
publisher |
MDPI AG |
series |
Genes |
issn |
2073-4425 |
publishDate |
2020-10-01 |
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′-<sup>32</sup>P]-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′-<sup>32</sup>P]-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. |
topic |
aminoacyl-tRNA synthetases prolyl-tRNA tyrosyl-tRNA biotinylation to aminoacyl-tRNA acp<sup>3</sup>U47 in tRNA |
url |
https://www.mdpi.com/2073-4425/11/10/1173 |
work_keys_str_mv |
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