In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity

Abstract

Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or "charging") specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. This activity relieves ambiguities during translation of the genetic code that result from one synthetase activating multiple amino acid substrates. In this review, we describe methods that have been developed for assaying both pre-and post-transfer editing activities. Pre-transfer editing is defined as hydrolysis of a misactivated aminoacyl-adenylate prior to transfer to the tRNA. This reaction has been reported to occur either in the aminoacylation active site or in a separate editing domain. Post-transfer editing refers to the hydrolysis reaction that cleaves the aminoacyl-ester linkage formed between the carbonyl carbon of the amino acid and the 2′ or 3′ hydroxyl group of the ribose on the terminal adenosine. Post-transfer editing takes place in a hydrolytic active site that is distinct from the site of amino acid activation. Here, we focus on methods for determination of steady-state reaction rates using editing assays developed for both classes of synthetases.

Coordination of tRNA Synthetase Active Sites for Chemical Fidelity

“…This would be expected based on the natural editing specificities of full-length IleRS and ValRS that would block isoleucine from the amino acid-binding pocket of the hydrolytic active site (10,29,30). Pyrophosphate exchange assays (19,24) support that isoleucine is activated by each of the hybrid M. mobile LeuRS proteins, albeit at a higher K m as compared with the cognate leucine substrate (Fig. 3 and supplemental Table S1).…”

“…Pyrophosphate Exchange-In a pyrophosphate exchange assay (20,24), K m and k cat for activation of leucine and also isoleucine, methionine, and valine were measured using equivalent 1 mM concentrations of amino acid, ATP, and [ 32 P]PP i (900 mCi/mmol) at 30°C. Pyrophosphate and ATP were separated by thin-layer chromatography and quantitated by phosphorimaging.…”

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

“…Traditional in vitro assays for determining aaRS editing activity use radiolabelled compounds [29]. These assays are labor intensive and are not practical for HTS.…”

“…Non-cognate aa are poor substrates for aaRSs and can exhibit a rate of activation for formation of the aminoacyl-adenylate intermediate that is 3 to 5 orders of magnitude lower than that of the cognate aa [31]. PPase in the coupled reaction helps to circumvent this problem because hydrolysis of PPi prevents reversal of the aa activation step and has been shown to drive aminoacylation forward for both cognate and non-cognate aa [29,32]. Thus, in this scheme, a drug targeting either of the aaRS's active sites (synthetic or editing) is predicted to decrease the kinetics of PPi synthesis.…”

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

“…Because 2′dA-tRNA and ddA-tRNA are missing the functional 2′ hydroxyl group that is necessary for aminoacylation to occur, AMP formation in these reactions would reflect cycles of amino acid activation and hydrolysis that are representative of pretransfer editing (31,32).…”