Mapping contacts between Escherichia coli alanyl tRNA synthetase and 2 ' hydroxyls using a complete tRNA molecule

A dual-specific derivative of yeast tRNA(Phe) is described whose features f acilitate structure-function studies of tRNAs. This tRNA has been made in t hree different bimolecular forms that allow modifications to be easily intr oduced into any position within the molecule. A set of deoxynucleotide subs tituted versions of this tRNA has been created and used to examine contacts between tRNA and Escherichia coli alanyl-tRNA synthetase, an enzyme previo usly shown to interact with 2'-hydroxyls in the acceptor stem of the tRNA. Because the present experiments used a full-length tRNA, several contacts w ere identified that had not been previously found using microhelix substrat es. Contacts at similar sites in the T-loop are seen in the cocrystal struc ture of tRNA(Ser) and Thermus thermophilus seryl-tRNA synthetase.