Expanding a tyrosyl-tRNA synthetase assay to other aminoacyl-tRNA synthetases.

Charles J Richardson,Eric A First

doi:10.1016/j.dib.2015.05.021

Charles J Richardson, Eric A First

Open Access

https://doi.org/10.1016/j.dib.2015.05.021

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Abstract

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. In general, aminoacyl-tRNA synthetase assays require stoichiometric amounts of tRNA, which limits their sensitivity while increasing their cost. This requirement for stoichiometric amounts of tRNA can be alleviated if the aminoacyl-tRNA product is cleaved following the tRNA aminoacylation reaction, regenerating the free tRNA substrate. This data article is related to the research article entitled “A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate” in which this approach is used to develop a continuous spectrophotometric assay for tyrosyl-tRNA synthetase [1]. Here we present enzymes that can be used to cleave the aminoacyl-tRNA product for at least 16 of the 20 naturally occurring amino acids. These enzymes can be used to extend the tyrosyl-tRNA synthetase assay to other aminoacyl-tRNA synthetases.

Highlights

Provides a blueprint for designing high-throughput assays for aminoacyl-tRNA synthetases Identifies editing domains and other proteins that can be used to regenerate free tRNA from the aminoacyl-tRNA product, decreasing the cost and increasing the sensitivity of aminoacyl-tRNA synthetase assays
We have developed a continuous spectrophotometric assay for one of the aminoacyl-tRNA synthetases, tyrosyl-tRNA synthetase, in which the release of AMP is coupled to the production of NADH via AMP deaminase and IMP dehydrogenase
We have demonstrated that the tyrosyl-tRNA synthetase assay can be used to monitor the aminoacylation of tRNA by either l- or d-tyrosine, with cyclodityrosine synthase and d-tyrosyl-tRNA deacylase being used to cleave the l-Tyr-tRNA and dTyr-tRNA products, respectively

Summary

Introduction

Expanding a tyrosyl-tRNA synthetase assay to other aminoacyl-tRNA synthetases Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. In general, aminoacyl-tRNA synthetase assays require stoichiometric amounts of tRNA, which limits their sensitivity while increasing their cost.

Results

Conclusion