Purification and Charging of Isoacceptor tRNAs

Yeast tRNA^Phe was purchased from Sigma-Aldrich. Other isoacceptor tRNAs were prepared from bulk tRNA (Roche) from either E. coli (tRNA^Val, tRNA^Lys, tRNA^Gln, and elongator tRNA^Met) or yeast (tRNA^Arg, tRNA^Trp, and tRNA^Leu) via hybridization to immobilized complementary oligoDNAs as described previously (1 (link), 39 (link)). E. coli and yeast tRNAs were charged with their cognate amino acids as described (56 (link), 57 (link)). E. coli tRNA^Gln was charged with either [³H]-Gln for the peptide release assay or [¹⁴C]-Gln for the Trp-tRNA^Trp(Prf) binding assay. Yeast tRNA^Trp was charged with [³H]-Trp for the hexapeptide assay or the hexapeptidyl-tRNA dissociation assay. E. coli elongator tRNA^Met was charged with [³⁵S]-Met for the octapeptide formation assay.

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Ng M.Y., Li H., Ghelfi M.D., Goldman Y.E, & Cooperman B.S. (2021). Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms. Proceedings of the National Academy of Sciences of the United States of America, 118(2), e2020599118.

Publication 2021

Yeast tRNAPhe

Amino acids Assay Bulk E coli Hybridization Peptide Trna Trnaarg Trnagln Trnaleu Trnalys Trnamet Trnaphe Trnatrp Trnaval Yeast

Corresponding Organization :

Other organizations : University of Pennsylvania

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Variable analysis

independent variables

Source of tRNA: Yeast or E. coli

dependent variables

Charged tRNA with specific labeled amino acids (3H-Gln, 14C-Gln, 3H-Trp, 35S-Met)

control variables

Yeast tRNA^Phe purchased from Sigma-Aldrich
Other tRNAs prepared from bulk tRNA (Roche) via hybridization to immobilized complementary oligoDNAs
Charging of tRNAs with cognate amino acids as described in cited references

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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