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A universal plate format for increased throughput of assays that monitor multiple aminoacyl transfer RNA synthetase activities.

ABSTRACT: Aminoacyl transfer RNA (tRNA) synthetases are intensely studied enzymes because of their importance in the establishment of the genetic code and their connection to disease and medicine. During the advancement of this field, several assays were developed. Despite many innovations, the sensitivity, simplicity, and reliability of the radiometric assays (which were among the first to be developed) have ensured their continued use. Four activities are measured by these assays: active site titration, amino acid activation, aminoacylation, and posttransfer editing (deacylation). In an effort to maintain the advantage of these assays while enhancing throughput, reducing waste, and improving data quality, a universal 96-well filter plate format was developed. This format facilitates the assays for all four of the widely studied activities.

SUBMITTER: Beebe K

PROVIDER: S-EPMC3833075 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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A universal plate format for increased throughput of assays that monitor multiple aminoacyl transfer RNA synthetase activities.

Beebe Kirk K Waas William W Druzina Zhanna Z Guo Min M Schimmel Paul P

Analytical biochemistry 20070518 1

Aminoacyl transfer RNA (tRNA) synthetases are intensely studied enzymes because of their importance in the establishment of the genetic code and their connection to disease and medicine. During the advancement of this field, several assays were developed. Despite many innovations, the sensitivity, simplicity, and reliability of the radiometric assays (which were among the first to be developed) have ensured their continued use. Four activities are measured by these assays: active site titration, ...[more]

PMID: 17603003

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Project description:Universal trees based on sequences of single gene homologs cannot be rooted. Iwabe et al. [Iwabe, N., Kuma, K.-I., Hasegawa, M., Osawa, S. & Miyata, T. (1989) Proc. Natl. Acad. Sci. USA 86, 9355-9359] circumvented this problem by using ancient gene duplications that predated the last common ancestor of all living things. Their separate, reciprocally rooted gene trees for elongation factors and ATPase subunits showed Bacteria (eubacteria) as branching first from the universal tree with Archaea (archaebacteria) and Eucarya (eukaryotes) as sister groups. Given its topical importance to evolutionary biology and concerns about the appropriateness of the ATPase data set, an evaluation of the universal tree root using other ancient gene duplications is essential. In this study, we derive a rooting for the universal tree using aminoacyl-tRNA synthetase genes, an extensive multigene family whose divergence likely preceded that of prokaryotes and eukaryotes. An approximately 1600-bp conserved region was sequenced from the isoleucyl-tRNA synthetases of several species representing deep evolutionary branches of eukaryotes (Nosema locustae), Bacteria (Aquifex pyrophilus and Thermotoga maritima) and Archaea (Pyrococcus furiosus and Sulfolobus acidocaldarius). In addition, a new valyl-tRNA synthetase was characterized from the protist Trichomonas vaginalis. Different phylogenetic methods were used to generate trees of isoleucyl-tRNA synthetases rooted by valyl- and leucyl-tRNA synthetases. All isoleucyl-tRNA synthetase trees showed Archaea and Eucarya as sister groups, providing strong confirmation for the universal tree rooting reported by Iwabe et al. As well, there was strong support for the monophyly (sensu Hennig) of Archaea. The valyl-tRNA synthetase gene from Tr. vaginalis clustered with other eukaryotic ValRS genes, which may have been transferred from the mitochondrial genome to the nuclear genome, suggesting that this amitochondrial trichomonad once harbored an endosymbiotic bacterium.

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A universal plate format for increased throughput of assays that monitor multiple aminoacyl transfer RNA synthetase activities.

Publications

A universal plate format for increased throughput of assays that monitor multiple aminoacyl transfer RNA synthetase activities.

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