Recognition of acceptor-stem structure of tRNA(Asp) by Escherichia coli aspartyl-tRNA synthetase

RNA. 2003 Apr;9(4):386-93. doi: 10.1261/rna.2139703.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aspartate-tRNA Ligase / metabolism*
  • Binding Sites
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Escherichia coli / growth & development
  • Protein Binding / physiology
  • RNA, Transfer, Asp / chemistry*
  • RNA, Transfer, Asp / metabolism*
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics


  • RNA, Transfer, Asp
  • Aspartate-tRNA Ligase