Evidence for Unfolding of the Single-Stranded GCCA 3‘-End of a tRNA on Its Aminoacyl-tRNA Synthetase from a Stacked Helical to a Foldback Conformation

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Abstract
The conformation of a tRNA in its initial contact with its cognate aminoacyl-tRNA synthetase was investigated with the Escherichiacoli glutamyl-tRNA synthetase−tRNAGlu complex. Covalent complexes between the periodate-oxidized tRNAGlu and its synthetase were obtained. These complexes are specific since none were formed with any other oxidized E.coli tRNA. The three major residues cross-linked to the 3‘-terminal adenosine of oxidized tRNAGlu are Lys115, Arg209, and Arg48. Modeling of the tRNAGlu−glutamyl-tRNA synthetase based on the known crystal structures of Thermusthermophilus GluRS and of the E.coli tRNAGln−glutaminyl-tRNA synthetase complex shows that these three residues are located in the pocket that binds the acceptor stem, and that Lys115, located in a 26 residue loop closed by coordination to a zinc atom in the tRNA acceptor stem-binding domain, is the first contact point of the 3‘-terminal adenosine of tRNAGlu. In our model, we assume that the 3‘-terminal GCCA single-stranded segment of tRNAGlu is helical and extends the stacking of the acceptor stem. This assumption is supported by the fact that the 3‘ CCA sequence of tRNAGlu is not readily circularized in the presence of T4 RNA ligase under conditions where several other tRNAs are circularized. The two other cross-linked sites are interpreted as the contact sites of the 3‘-terminal ribose on the enzyme during the unfolding and movement of the 3‘-terminal GCCA segment to position the acceptor ribose in the catalytic site for aminoacylation.