Proofreading by an aminoacyl-tRNA synthetase. (a) tRNA^Val is charged in the acylation site of the Val-tRNA synthetase. Isoleucine is larger than valine and does not fit in the acylation site. (b) tRNA^Ile is charged in the acylation site of the Ile-tRNA synthetase. Because valine is smaller than isoleucine, tRNA^Ile is sometimes charged with valine. This incorrectly charged Val-tRNA^Ile fits into the synthetase’s proofreading site, where it is hydrolyzed to release valine from the tRNA.