Preparation and evaluation of acylated tRNAs.

In the cell, the activity of tRNA is governed by its acylation state. Interactions with the ribosome, translation factors, and regulatory elements are strongly influenced by the acyl group, and presumably other cellular components that interact with tRNA also use the acyl group as a specificity determinant. Thus, those using biochemical approaches to study any aspect of tRNA biology should be familiar with effective methods to prepare and evaluate acylated tRNA reagents. Here, methods to prepare aminoacyl-tRNA, N-acetyl-aminoacyl-tRNA, and fMet-tRNA(fMet) and to assess their homogeneity are described. Using these methods, acylated tRNAs of high homogeneity can be reliably obtained.