A Label-Free Assay for Aminoacylation of tRNA

Abstract

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.

Genome-Wide Profiling of tRNA Using an Unexplored Reverse Transcriptase with High Processivity

“…The plasmid was then introduced into bacteria by transformation, purified, and subsequently introduced into the S. cerevisiae BY4741 strain by transformation. Expression of each human tRNA gene in yeast was verified using a label-free assay for aminoacylation of tRNA as described 78 .…”

Starvation sensing by mycobacterial RelA/SpoT homologue through constitutive surveillance of translation

“…The charging reaction (2 mL) contained approximately 200 nmoles of total tRNA and 10 nmoles of MetRS in the presence of 1 mM methionine, 2 mM ATP, 10 mM MgCl 2 , 20 mM KCl, 4 mM DTT, 50 μg/mL BSA, and 50 mM Tris-HCl pH 7.5. After 10 min at 37 °C a 4.0 μL aliquot of the reaction was removed for determination of the charging efficiency as described by Gamper et al (Gamper and Hou, 2020). The remaining reaction was incubated for 10 min at 37 °C with 1.7 µmoles of 10-formyltetrahydrofolate and 20 nmoles of methionyl formyl transferase to convert Met-tRNA i Met to fMet-tRNA i Met .…”

Twice exploration of tRNA +1 frameshifting in an elongation cycle of protein synthesis

“…The control culture had cells harboring a lacZ reporter plasmid and the empty pKK223-3 plasmid and were induced with 0.4 mM IPTG at 37°C for 4 h. Cells were harvested and an aliquot of the cell lysate was assayed for the frequency of +1 frameshifting by measuring the lacZ activity in cells expressing the CCC-C reporter plasmid relative to cells expressing the CCC control plasmid ( 13 ). A separate aliquot of the cell lysate was quantified for tRNA expression in a label-free aminoacylation assay ( 28 ), in which total tRNA from cell lysates over-expressing hisT or thrV was aminoacylated by the respective E. coli HisRS and ThrRS, biotinylated with sulfo-NHS-biotin, ethanol precipitated twice, and bound to streptavidin. Charged aa-tRNA was separated from uncharged tRNA by a denaturing 12% PAGE/7 M urea gel and quantified by SYBR Gold.…”