Exploring the Role in Translation Quality Control of Cytoplasmic Phenylalanyl-tRNA Synthetase
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Date
2010-06
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The Ohio State University
Abstract
A critical step in gene expression is the faithful translation of protein from messenger RNA. This process relies on the attachment of specifically paired amino acids to their respective tRNAs by aminoacyl-tRNA synthetases (aaRSs). To ensure high accuracy of tRNA aminoacylation, aaRSs rely on maintaining high substrate specificity and editing activity. While editing significantly decreases the frequency of errors during aa-tRNA synthesis in vitro, many details of the reaction remain unknown. Eukaryotic organisms contain two distinct phenylalanyl-tRNA synthetases (PheRS), a cytoplasmic (cytoPheRS) and a mitochondrial form (mitotPheRS). Saccharomyces cerevisiae cytoPheRS has lost some amino acid specificity but maintains an editing acitivity, while the mitoPheRS maintains amino acid specificity but has lost editing activity. To investigate the impact of editing activity on the cell, S. cerevisiae cytoPheRS was engineered to have a high level of amino acid specifity with and without editing activity. To do this the wild-type FRS2 gene, which encodes the cytoPheRS α-subunit and contains the active site, was cloned onto a centromeric plasmid and used to complement a heterozygous FRS2 deletion (FRS2/frs2D). The FRS2 wild-type gene product was found to be functional in vivo. The FRS2 specificity mutant (frs2-1) was also cloned and determined to be functional in vivo as well. The functional frs2-1 strain will next be used to complement an editing defective strain (frs1-1) which shows an amino acid dependent growth defect. This work will help clarify the impact of amino acid specificity and post-transfer editing on cellular physiology.
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Keywords
aminoacyl-tRNA synthetase, protein synthesis, cytoPheRS, tRNA
Citation
Cell-specific differences in the requirements for translation quality control PNAS 2010 107 (9) 4063-4068