Poster abstracts
Poster number 59 submitted by Bradley Howard
Prolyl-tRNA synthetase–like editing domains: Exploring cellular functions in E. coli.
C. Bradley Howard (Department of Chemistry and Biochemistry and the Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Mom Das (Department of Chemistry and Biochemistry and the Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry and the Center for RNA Biology, The Ohio State University, Columbus, OH 43210)
Abstract:
Despite the importance of protein fidelity in translation for proper cellular function, prolyl-tRNA synthetase (ProRS) has been shown to mischarge amino acids Ala and Cys, which are smaller or similar in size to cognate Pro. Fidelity is maintained via tRNA editing. In most bacteria, the Ala-specific editing activity resides in the INS domain of ProRS, whereas YbaK, a single domain trans-editing INS homolog, is responsible for Cys-tRNAPro deacylation. Four other INS homologs (ProXp-ala, ProXp-ST1, ProXp-ST2, and ProXp-abu) have been identified and shown to have in vitro deacylation activity.The physiological roles of INS homologs have not been characterized in vivo. Therefore, the goal of this work is to identify potential cellular functions and pathways in which INS homologs participate. Published transcriptomic data correlates the expression of these proteins with cellular stresses (e.g., oxidative, heat, and cold). Using the comparative genomic database SEED, these homologs cluster in many bacteria with periplasmic proteins and amino acid metabolic proteins, suggesting periplasmic localization and potentially the regulation of amino acid levels. INS homologs are common among metabolically diverse bacteria, including soil bacteria and pathogens. Tandem-affinity chromatography in E. coli has linked YbaK and ProXp-ST1 with glycolysis, amino acid biosynthesis, translation, and aminoacylation. Amino acid misincorporation will be examined in YbaK and ProXp-ST1 knockout strains under a variety of stress conditions using a gain-of-activity misincorporation GFP reporter. In a parallel approach, mistranslation levels will be measured via mass spectrometry analysis of a Pro-rich protein expressed in the null strains. Since ProRS-like trans-editing domains are not present in higher eukaryotes including humans, identifying the conditions in which these proteins are essential may lead to new avenues for antibiotic development.
Keywords: aminoacyl-tRNA synthetases, tRNA editing, proline