Recoding Aminoacyl-tRNA Synthetases for Synthetic Biology by Rational Protein-RNA Engineering

We have taken a rational approach to redesigning the amino acid binding and aminoacyltRNA pairing specificities of bacterial glutaminyltRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 105-fold compared to the wild-type enzyme. Better optimized designs of the proteinRNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyltRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.