Mapping Contacts between Escherichia coli Alanyl tRNA Synthetase and 2‘ Hydroxyls Using a Complete tRNA Molecule

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Abstract
A dual-specific derivative of yeast tRNAPhe is described whose features facilitate structure−function studies of tRNAs. This tRNA has been made in three different bimolecular forms that allow modifications to be easily introduced into any position within the molecule. A set of deoxynucleotide substituted versions of this tRNA has been created and used to examine contacts between tRNA and Escherichia coli alanyl-tRNA synthetase, an enzyme previously shown to interact with 2‘-hydroxyls in the acceptor stem of the tRNA. Because the present experiments used a full-length tRNA, several contacts were identified that had not been previously found using microhelix substrates. Contacts at similar sites in the T-loop are seen in the cocrystal structure of tRNASer and Thermus thermophilus seryl-tRNA synthetase.