Synthesis of Glu-tRNA^Gln by Engineered and Natural Aminoacyl-tRNA Synthetases

Abstract

A protein engineering approach is described to delineate which distinct elements of homologous tRNA synthetase architectures are responsible for divergent RNA-amino acid pairing specificities. Previously, we constructed a hybrid enzyme in which 23 amino acids from the catalytic domain of E. coli glutaminyl-tRNA synthetase (GlnRS) were replaced with the corresponding residues of human glutamyl-tRNA synthetase (GluRS). The engineered hybrid (GlnRS S1/L1/L2) synthesizes Glu-tRNAGln over 104-fold more efficiently than GlnRS. Detailed comparison of kinetic parameters between GlnRS S1/L1/L2 and the naturally occurring M. thermautotrophicus GluRSND, which is also capable of Glu-tRNAGln synthesis, now shows that both kcat and Km for glutamate are recapitulated in the engineered enzyme, but that Km for tRNA is 200-fold higher. Thus, the simultaneous optimization of paired amino acid and tRNA binding sites found in a naturally occurring enzyme is not recapitulated in a hybrid that is successfully engineered for amino acid complementarity. We infer that the GlnRS architecture has differentiated to match only cognate amino acid-RNA pairs, and that the substrate selection functions do not operate independently of each other. Design and characterization of four additional hybrids identifies further residues involved in improving complementarity for glutamate, and in communicating between amino acid and tRNA binding sites. The robust catalytic function demonstrated in this engineered system offers a novel platform for exploring the stereochemical origins of coding as a property of the ancient Rossmann fold.

Rational design of an evolutionary precursor of glutaminyl-tRNA synthetase

“…Although in some cases lacking base modifications can lead to inactive tRNAs (10), the kinetic values measured for WT-GluRS are within the typical range for aaRSs and indicate the role of modified bases is not critical for WT-GluRS activity. The kinetic constants of WTGluRS for in vitro transcribed tRNA Gln2 were recently measured (13), showing the enzyme to be marginally (8.5 AE 3.6-fold) less efficient in our experiments.…”

Kinetics of tRNA folding monitored by aminoacylation

“…2B). The rate constant for the burst phase is about threefold lower than the k max previously measured for single-turnover aminoacylation reactions (6 min À1 ) (Rodriguez-Hernandez et al 2010). This is attributable to the lower temperature of the reaction (21°C instead of 37°C) and the subsaturating concentration of GluRS ND (100 nM).…”

“…Previously, we determined k cat , K m (tRNA Gln (CUG) ), K m (Glu), and K m (ATP) for Glu-tRNA Gln synthesis by the nondiscriminating glutamyl-tRNA synthetase (GluRS ND ) from Methanothermobacter thermautotrophicus (MT) (Rodriguez-Hernandez et al 2010). Maximal plateau levels for aminoacylation were obtained by folding the unmodified 32 P-labeled tRNA Gln transcript by heating to 80°C, adding MgCl 2 to a final concentration of 10 mM, and slow-cooling to the ambient temperature of 21°C (Fig.…”

“…Oligonucleotide design and duplex DNA template synthesis for the MT tRNA Gln (CUG) were performed as described (Rodriguez-Hernandez et al 2010). To synthesize the MT tRNA, 5 mg/mL duplex DNA template in a solution containing 40 mM Tris-HCl (pH 8.0), 25 mM MgCl 2 , 2 mM spermidine, 0.01% Triton-x, 40 mM DTT, and 1 mM each of ATP, GTP, CTP, and UTP was incubated with 120 mg/mL T7 RNA polymerase in a 5 mL reaction for 4 h at 37°C.…”

“…Preparation of 39-32 P-labeled tRNAs via the exchange reaction of tRNA nucleotidyltransferase was performed as described (Ledoux and Uhlenbeck 2008;Rodriguez-Hernandez et al 2010;RodriguezHernandez and Perona 2011). All aminoacylation reactions were performed at 100 mM Na-Hepes (pH 7.0), 10 mM MgCl 2 2+ , and 5 mM DTT unless otherwise noted.…”