Rational design of an evolutionary precursor of glutaminyl-tRNA synthetase

Abstract

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 Gln2CUG. The less active tRNA Gln1UUG 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 Gln2CUG isoacceptor, which responds to the CAG codon, and shows no activity toward tRNA Gln1UUG. 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.