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  • br Materials and methods br Data availab http

    2018-10-23


    Materials and methods
    Data availability The mass spectrometry datasets are publicly available from ProteomeXchange Consortium via the PRIDE partner repository (http://www.proteomexchange.org) with the dataset identifiers PXD001224 (GeLC–MSMS data) and PXD001088 (LC–MSMS data). Results obtained from mass spectrometry post MaxQuant analysis are available as Supplementary materials with this paper. A list of different types of data made available by this paper is presented as Table 1.
    Funding This work has been supported by The Netherlands Organization for Scientific Research (NWO) supported large-scale proteomics facility Proteins At Work (project 184.032.201) embedded in the Netherlands Proteomics Centre and by the PRIME-XS project grant agreement number 262067 supported by the European Community׳s Seventh Framework Programme (FP7/2007-2013) to AJRH.
    Acknowledgments
    Specifications table
    Value of the data
    Data, experimental design, materials and methods
    Specifications table
    Data, experimental design, materials and methods Aminoacyl-tRNA synthetases (aaRSes) are essential enzymes that catalyze the attachment of amino acids to their cognate tRNAs using a two-step mechanism (Fig. 1). In the first step, the amino beta amyloid is activated by ATP, forming an enzyme-bound aminoacyl-adenylate intermediate (aaRS•AA-AMP). In the second step of the reaction, the activated aminoacyl-moiety is transferred to the 3׳ end of the cognate tRNA, resulting in the release of the aminoacyl-tRNA and AMP products. 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 (which converts AMP to IMP) and IMP dehydrogenase (which couples the reduction of NAD+ to the conversion of IMP to XMP). As the production of NADH is associated with an increase in absorbance at 340nm, the aminoacylation of tRNATyr by tyrosine can be monitored spectrophotometrically. In contrast to other aminoacyl-tRNA synthetase assays, where tRNA is the limiting substrate, in the tyrosyl-tRNA synthetase assay, the Tyr-tRNATyr product is cleaved, regenerating the tRNATyr substrate. This results in a substantial increase in the sensitivity of the assay, while significantly decreasing its cost. 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 d-Tyr-tRNA products, respectively. A detailed description of this assay can be found in [1]. The basic aminoacyl-tRNA synthetase assay is shown in Fig. 2. Aminoacylation of tRNA results in the release of the aminoacyl-tRNA product, AMP, and inorganic pyrophosphate (PPi). We have coupled the production of AMP to the reduction of NAD+, allowing the assay to be followed by monitoring changes in absorbance at 340nm (ε340NADH=6220M−1cm−1). Alternatively, the reaction can be followed by using inorganic pyrophosphatase to cleave the inorganic pyrophosphate product and monitoring the resulting production of phosphate (e.g. via reaction with malachite green and ammonium molybdate [2,3]). Cleavage of the aminoacyl-tRNA product is achieved by using an editing domain, trans-editing protein, or cyclodipeptide synthase that is specific for each particular aminoacyl-tRNA, or by using the M129K variant of d-tyrosyl-tRNA deacylase, which is proposed to catalyze the hydrolysis of both l- and d-aminoacyl-tRNAs and has a broad specificity with respect to the aminoacyl moiety (Table 1). In the event that a particular editing domain cannot be isolated in its active form, a variant of the full length aminoacyl-tRNA synthetase in which the synthetic site has been inactivated can be used to hydrolyze the aminoacyl-tRNA product.