Breaking the Stereo Barrier of Amino Acid Attachment to tRNA by a Single Nucleotide

Aminoacyl-tRNA synthetases are responsible for attaching amino acid residues to the tRNA 3′-end. The two classes of synthetases approach tRNA as mirror images, with opposite but symmetrical stereochemistries that allow the class I enzymes to attach amino acid residues to the 2′-hydroxyl group of the terminal ribose, whereas, the class II enzymes attach amino acid residues to the 3′-hydroxyl group. However, we show here that the attachment of cysteine to tRNACys by the class I cysteinyl-tRNA synthetase (CysRS) is flexible; the enzyme is capable of using either the 2′ or 3′-hydroxyl group as the attachment site. The molecular basis for this flexibility was investigated. Introduction of the nucleotide U73 of tRNACys into tRNAVal was found to confer the flexibility. While valylation of the wild-type tRNAVal by the class I ValRS was strictly dependent on the terminal 2′-hydroxyl group, that of the U73 mutant of tRNAVal occurred at either the 2′ or 3′-hydroxyl group. Thus, the single nucleotide U73 of tRNA has the ability to break the stereo barrier of amino acid attachment to tRNA, by mobilizing the 2′ and 3′-hydroxyl groups of A76 in flexible geometry with respect to the tRNA acceptor stem.