Recoding Aminoacyl-tRNA Synthetases for Synthetic
Biology by Rational Protein-RNA Engineering
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Abstract
We
have taken a rational approach to redesigning the amino acid
binding and aminoacyl–tRNA pairing specificities of bacterial
glutaminyl–tRNA synthetase. The four-stage engineering incorporates
generalizable design principles and improves the pairing efficiency
of noncognate glutamate with tRNA<sup>Gln</sup> by over 10<sup>5</sup>-fold compared to the wild-type enzyme. Better optimized designs
of the protein–RNA 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 aminoacyl–tRNA synthetases that ligate noncanonical
amino acids onto tRNAs. This will facilitate reconstruction of the
cellular translation apparatus for applications in synthetic biology