A NEW ASSAY FOR TRANSFER-RNA AMINOACYLATION KINETICS

An improved quantitative assay for tRNA aminoacylation is presented ba sed on charging of a nicked tRNA followed by separation of an aminoacy lated 3'-fragment on an acidic denaturing polyacrylamide gel. Kinetic parameters of tRNA aminoacylation by Escherichia coli AlaRS obtained b y the new method are in excellent agreement with those measured by the conventional method. This assay provides several advantages over the traditional methods of measuring tRNA aminoacylation: (1) the fraction of aminoacyl-tRNA is measured directly; (2) data can be obtained at s aturating amino acid concentrations; and (3) the assay is significantl y more sensitive.