Poster abstracts
Poster number 39 submitted by Bradley Howard
Probing the physiological function of a prolyl-tRNA synthetase-related trans-editing domain
C. Bradley Howard (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, Ohio), Ziwei Liu (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, Ohio), Karin Musier-Forsyth (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, Ohio)
Abstract:
Although organisms can tolerate some mistakes in translation without a substantial fitness cost, cellular conditions that increase the rate of mistranslation above this threshold can result in toxicity in bacteria and pathologies in mammalian cells. Aminoacyl-tRNA synthetases (ARSs) attach each amino acid (aa) to its cognate tRNA, but errors can occur in the presence of similar aa’s. About half of the ARSs have been shown to possess proofreading capabilities in vitro, which clear errors before (pre-) or after (post-transfer editing) the formation of aa-tRNA. Post-transfer editing may occur in cis (via specialized domains appended to the ARS) or in trans (via free-standing editing domains). We have identified a family of trans-editing factors homologous to the bacterial prolyl-tRNA synthetase cis-editing domain (INS), including ProXp-ST1 in E. coli. This trans-editing factor has been shown to edit Ser/Thr-tRNA in vitro, but this activity has not been demonstrated in vivo. Although deletion of these factors does not result in a growth defect under normal conditions, we hypothesize that trans editing domains become critical under conditions where the balance of aa’s and aa-like metabolites is perturbed, generating mischarged aa-tRNAs and an altered protein pool. To test this hypothesis and to verify the substrate(s) of ProXp-ST1, we have developed a gain-of-function reporter system for measuring misincorporation at several codons in a deletion strain. The critical Ser80 of beta-lactamase is replaced with several codons (whose ARSs are known to mischarge Ser onto their tRNA), thus inactivating the enzyme. Only the misincorporation of Ser will rescue growth in the presence of ampicillin, which will be exacerbated in the deletion strain if ProXP-ST1 edits Ser-tRNAs. The misincorporation events will also be validated by mass spectrometry. Preliminary data demonstrating an in vivo substrate of ProXp-ST1 will be presented.
References:
Liu, Z. et al. Proc Natl Acad Sci U S A 2015, 112 (19), 6027-32.
Keywords: aminoacyl tRNA synthetase, trans editing, misincorporation