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Proc Natl Acad Sci U S A. 2011 Dec 20;108(51):20485-90. doi: 10.1073/pnas.1117294108. Epub 2011 Dec 7.

Rational design of an evolutionary precursor of glutaminyl-tRNA synthetase.

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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.


The specificity of most aminoacyl-tRNA synthetases for an amino acid and cognate tRNA pair evolved before the divergence of the three domains of life. Glutaminyl-tRNA synthetase (GlnRS) evolved later and is derived from the archaeal-type nondiscriminating glutamyl-tRNA synthetase (GluRS), an enzyme with relaxed tRNA specificity capable of forming both Glu-tRNA(Glu) and Glu-tRNA(Gln). The archaea lack GlnRS and use a specialized amidotransferase to convert Glu-tRNA(Gln) to Gln-tRNA(Gln) needed for protein synthesis. We show that the Methanothermobacter thermautotrophicus GluRS is active toward tRNA(Glu) and the two tRNA(Gln) isoacceptors the organism encodes, but with a significant catalytic preference for tRNA(Gln2)(CUG). The less active tRNA(Gln1)(UUG) responds to the less common CAA codon for Gln. From a biochemical characterization of M. thermautotrophicus GluRS variants, we found that the evolution of tRNA specificity in GlnRS could be recapitulated by converting the M. thermautotrophicus GluRS to a tRNA(Gln) specific enzyme, solely through the addition of an acceptor stem loop present in bacterial GlnRS. One designed GluRS variant is also highly specific for the tRNA(Gln2)(CUG) isoacceptor, which responds to the CAG codon, and shows no activity toward tRNA(Gln1)(UUG). Because it is now possible to eliminate particular codons from the genome of Escherichia coli, additional codons will become available for genetic code engineering. Isoacceptor-specific aminoacyl-tRNA synthetases will enable the reassignment of more open codons while preserving accurate encoding of the 20 canonical amino acids.

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