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

Near‐cognate suppression of amber, opal and quadruplet codons competes with aminoacyl‐tRNAPyl for genetic code expansion

“…pETtrio-pylT(CUA)-PylRS-sfGFP134TAG [24] (K.A.O. & W.R.L unpublished data) was quick-changed to pETtrio-pylT( UCCU)-sfGFP134AGGA, encoding tRNA Pyl UCCU and a C-terminal His 6 -tagged superfolder green fluorescent protein (sfGFP) with AGGA at codon 134. tRNAs were cloned into pUC18 ( Xba I/ BamH I) for in vitro transcription, which was performed as described [25]. His 6 -tagged PylRS (in pet15b) was over-expressed and purified as before [26].…”

“…Aminoacylation assays were performed for all AARS/tRNA pairs as described [25]. Enzyme, tRNA, amino acid, and ATP were included in excess concentrations to detect low activity reactions.…”

Neuronal protection by a variant of GAPDH pseudogene P44 in AD

“…We explored the biological consequence of the SNP, which results in a codon change from CAG to CAA. Both of these codons code for glutamine, however, the literature suggests that the fidelity of this incorporation is not absolute [26,27]. Therefore, we propose that a glutamate residue may be inserted in place of a glutamine residue, particularly in times nutritional compromise.…”

Engineering oxidoreductases: maquette proteins designed from scratch

“…This is particularly apparent in the growing literature of enzymes with substrate specificity rationally altered, usually by changing active-site loops [10,11]. Beyond substrate recognition and enzymatically competent binding, much recent progress has been made in wholescale modification or generation of catalytic activity in protein scaffolds [12–14]. The Baker group has led the way in producing enzymatic function built into natural scaffolds, including the creation of enzymes capable of catalysing reactions not known to occur naturally [15,16].…”