Anticodon recognition in evolution - Switching tRNA specificity of an aminoacyl-tRNA synthetase by site-directed peptide transplantation

The highly conserved aspartyl-, asparaginyl-, and lysyl-tRNA synthetases compose one subclass of amino-acyl-tRNA synthetases, called IIb. The three enzymes possess an OB-folded extension at their N terminus. The function of this extension is to specifically recognize the anticodon triplet of the tRNA. Three-dimensional models of bacterial aspartyl- and lysyl-tRNA synthetases complexed to tRNA indicate that a rigid scaffold of amino acid residues along the five beta-strands of the OB-fold accommodates the base U at the center of the anticodon. The binding of the adjacent anticodon bases occurs through interactions with a flexible loop joining strands 4 and 5 (L(45)). As a result, a switching of the specificity of lysyl-tRNA synthetase from tRNA(Lys) (anticodon UUU) toward tRNA(Asp) (GUC) could be attempted by transplanting the small loop L(45) of aspartyl- tRNA synthetase inside lysyl-tRNA synthetase. Upon this transplantation, lysyl-tRNA synthetase loses its capacity to aminoacylate tRNA(Lys). In exchange, the chimeric enzyme acquires the capacity to charge tRNA(Asp) with lysine. Upon giving the tRNA(Asp) substrate the discriminator base of tRNA(Lys), the specificity shift is improved. The change of specificity was also established in vivo. Indeed, the transplanted lysyl-tRNA synthetase succeeds in suppressing a missense Lys --> Asp mutation inserted into the beta-lactamase gene. These results functionally establish that sequence variation in a small peptide region of subclass IIb aminoacyl-tRNA synthetases contributes to specification of nucleic acid recognition. Because this peptide element is not part of the core catalytic structure, it may have evolved independently of the active sites of these synthetases.