A Label-Free Assay for Aminoacylation of tRNA

• Main Authors: Howard Gamper, Ya-Ming Hou
• Format: Article
• Language: English
• Published: MDPI AG 2020-10-01
• Series: Genes
• Subjects: aminoacyl-tRNA synthetases; prolyl-tRNA; tyrosyl-tRNA; biotinylation to aminoacyl-tRNA; acp³U47 in tRNA
• Online Access: https://www.mdpi.com/2073-4425/11/10/1173

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.