Recognition of acceptor-stem structure of tRNAAsp by Escherichia coli aspartyl-tRNA synthetase

Protein–RNA recognition between aminoacyl-tRNA synthetases and tRNA is highly specific and essential for cell viability. We investigated the structure–function relationships involved in the interaction of the Escherichia coli tRNA Asp acceptor stem with aspartyl-tRNA synthetase. The goal was to isolate functionally active mutants and interpret them in terms of the crystal structure of the synthetase-tRNA Asp complex. Mutants were derived from Saccharomyces cerevisiae tRNA Asp , which is inactive with E. coli aspartyl-tRNA synthetase, allowing a genetic selection of active tRNAs in a tRNA Asp knockout strain of E. coli . The mutants were obtained by directed mutagenesis or library selections that targeted the acceptor stem of the yeast tRNA Asp gene. The mutants provide a rich source of tRNA Asp sequences, which show that the sequence of the acceptor stem can be extensively altered while allowing the tRNA to retain substantial aminoacylation and cell-growth functions. The predominance of tRNA backbone-mediated interactions observed between the synthetase and the acceptor stem of the tRNA in the crystal and the mutability of the acceptor stem suggest that many of the corresponding wild-type bases are replaceable by alternative sequences, so long as they preserve the initial backbone structure of the tRNA. Backbone interactions emerge as an important functional component of the tRNA-synthetase interaction.