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

The conformation of a tRNA in its initial contact with its cognate aminoacy l-tRNA synthetase was investigated with the Escherichia coli glutamyl-tRNA synthetase-tRNA(Glu) complex. Covalent complexes between the periodate-oxid ized tRNA(Glu) and its synthetase were obtained. These complexes are specif ic since none were formed with any other oxidized E. coli tRNA. The three m ajor residues cross-linked to the 3'-terminal adenosine of oxidized tRNA(Gl u) are Lys115, Arg209, and Arg48. Modeling of the tRNA(Glu)-glutamyl-tRNA s ynthetase based on the known crystal structures of Thermus thermophilus Glu RS and of the E. coli tRNA(Gln)-glutaminyl-tRNA synthetase complex shows th at these three residues are located in the pocket that binds the acceptor s tem, and that Lys115, located in a 26 residue loop closed by coordination t o a zinc atom in the tRNA acceptor stem-binding domain, is the first contac t point of the 3'-terminal adenosine of tRNA(Glu). In our model, we assume that the 3'-terminal GCCA single-stranded segment of tRNA(Glu) is helical a nd extends the stacking of the acceptor stem. This assumption is supported by the fact that the 3' CCA sequence of tRNA(Glu) is not readily circulariz ed in the presence of T4 RNA ligase under conditions where several other tR NAs are circularized. The two other cross-linked sites are interpreted as t he contact sites of the 3'-terminal ribose on the enzyme during the unfoldi ng and movement of the 3'-terminal GCCA segment to position the acceptor ri bose in the catalytic site for aminoacylation.