Partitioning of tRNA-dependent Editing between Pre- and Post-transfer Pathways in Class I Aminoacyl-tRNA Synthetases

Abstract

Hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes occurs in a spatially separate domain inserted into the catalytic Rossmann fold, but the location and mechanisms of pre-transfer hydrolysis of misactivated amino acids have been uncertain. Here, we use novel kinetic approaches to distinguish among three models for pre-transfer editing by Escherichia coli isoleucyl-tRNA synthetase (IleRS). We demonstrate that tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme and that noncatalytic hydrolysis after release is too slow to account for the observed rate of clearing. Measurements of the microscopic rate constants for amino acid transfer to tRNA in IleRS and the related valyl-tRNA synthetase (ValRS) further suggest that pre-transfer editing in IleRS is an enzyme-catalyzed activity residing in the synthetic active site. In this model, the balance between pretransfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate. Rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS, whereas in ValRS transfer to tRNA is 200-fold faster than hydrolysis. In consequence, editing by ValRS occurs nearly exclusively by post-transfer hydrolysis in the editing domain, whereas in IleRS both pre-and post-transfer editing are important. In both enzymes, the rates of amino acid transfer to tRNA are similar for cognate and noncognate aminoacyl-adenylates, providing a significant contrast with editing DNA polymerases.

Kinetics of tRNA folding monitored by aminoacylation

“…This assay possesses significant advantages over the conventional method that relies instead on radiolabeled amino acid: (1) The fraction of aminoacylable tRNA (plateau level) can be directly monitored by the ratio of substrate and product intensities on a thin-layer chromatography (TLC) plate; (2) the sensitivity of the assay is much higher, enabling the detection of even very weak aminoacylation levels; and (3) the use of unlabeled amino acid allows high and saturating concentrations of this substrate to be used. This assay is generally applicable to many and perhaps all aaRSs, including those that utilize nonstandard amino acid substrates, and yields precise measurements of steady-state and elementary rate constants for both cognate and misacylation reactions (Uter and Perona 2004;Hauenstein et al 2008;Ledoux and Uhlenbeck 2008;Dulic et al 2010). We describe now the further use of this assay to monitor precatalytic folding of the tRNA substrate, including the influence of posttranscriptional modifications on this process.…”

Substrate-mediated Fidelity Mechanism Ensures Accurate Decoding of Proline Codons

“…In this case, a coordinated zinc ion in the editing site of AlaRS may help provide specificity for non-cognate serine, which is larger than cognate alanine (20,21). Insights into the relative contributions of pre-and post-transfer editing have also begun to emerge in several Class I and Class II synthetase systems (22)(23)(24)(25)(26).Due to its unique side chain with special chemical properties, cysteine should be relatively easy for synthetases to discriminate. Similarly, proline is the only imino acid and would not be predicted to be especially problematic.…”

Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

“…A preponderance of posttransfer editing versus pretransfer editing can be dictated by a rapid aminoacyl-transfer step in the aminoacylation site (22,23,41). Likewise, the CP1 domain in IleRS relies upon pretransfer editing as its dominant editing pathway (42) because of a slow transfer rate (22).…”

“…Both tRNA-dependent (20)(21)(22)(23) and tRNA-independent (22, 24) pretransfer editing activities have been reported. Also, clearance of the adenylate by selective ejection from the enzyme's active site into the aqueous environment has been suggested (24).…”