Hypersaline environments contain a diverse range of halophilic organisms such as the archaeal species, Halobacterium salinarum (Hs). This organism, which lives at saturating salt concentrations, has been found to adapt to this environment by matching the extracellular ionic concentration with intracellular salts. Because of the large amount of intracellular salts, its enzymes are adapted to function under high ionic conditions. Hs cysteinyl-tRNA synthetase (Hs CysRS) is an enzyme that catalyzes the attachment of cysteine to its cognate tRNACys under high salt concentrations. Due to its activity only under high salt concentrations, quantifying the enzymatic activity of the Hs CysRS is difficult. To circumvent this problem and normalize the aminoacylation assay, we are using capillary gel electrophoresis (CGE) to determine the quantity and quality of the cysteinyl-tRNACys formed from the reaction of CysRS, tRNACys and cysteine. These results will be confirmed by mass spectrometry and gel-shift assays. Once it is determined CGE can be used to quantitate the aminoacylated products, we can embark on a mutational analysis to study the halophilic nature of this enzyme and its tRNA.

Development of a method to separate aminoacylated tRNA using capillary gel electrophoresis

ACS Spring 2022

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