[3′-³²P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation

The analysis of reactions involving amino acids esterified to tRNAs traditionally uses radiolabeled amino acids. We describe here an alternative assay involving [3′-³²P]-labeled tRNA followed by nuclease digestion and TLC analysis that permits aminoacylation to be monitored in an efficient, quantitative manner while circumventing many of the problems faced when using radiolabeled amino acids. We also describe a similar assay using [3′-³²P]-labeled aa-tRNAs to determine the rate of peptide bond formation on the ribosome. This type of assay can also potentially be adapted to study other reactions involving an amino acid or peptide esterified to tRNA.