Poster abstracts
Poster number 20 submitted by Nathan Kudlapur
Characterization of glutamyl-tRNA synthetase point mutations implicated in human disease
Nathan Kudlapur (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Danni Jin (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)
Abstract:
Aminoacyl-tRNA synthetases (AARS) are a class of enzymes that catalyze the charging of tRNAs with cognate amino acids, a critical step that contributes to the fidelity of protein synthesis. Many AARSs also possess non-canonical functions such as regulation of apoptosis, mRNA translation, and RNA splicing. Some AARSs have evolved new domains with no apparent connection to their charging functions. For example, WHEP domains were originally identified in tryptophanyl-tRNA synthetase (WRS), histidyl-tRNA synthetase (HRS), and glutamyl-prolyl-tRNA synthetase (EPRS). EPRS is a unique bifunctional AARS, found only in higher eukaryotic systems, that consists of two synthetase domains—glutamyl-tRNA synthetase (ERS) and prolyl-tRNA synthetase (PRS)—joined by a non-catalytic linker region containing three WHEP domains in humans. Two point mutations within the ERS portion of human EPRS (P14R and E205G) have been identified in the genomes of two patients with diabetes and bone disease. However, the mechanism by which these compound heterozygous mutations contribute to disease is unknown. The overall goal of this project is to determine whether the point mutations affect the catalytic activity of EPRS responsible for tRNA charging or non-canonical functions. In this work, we are investigating the enzymatic activities of wild-type and mutant recombinant ERS constructs, overexpressed and purified from Escherichia coli, containing varying portions of the linker (WHEP) domain. We successfully purified two wild-type ERS constructs: a 749 amino acid (aa) construct containing the ERS domain and a short random coil segment of the linker, and a 929aa construct containing ERS in addition to 2.5 WHEP domains. Binding assays indicate the 929aa construct has stronger tRNAGlu binding affinity than the 749aa construct. Aminoacylation assays have revealed that both constructs are active in tRNAGlu charging, with similar reaction rates. The two individual point mutants and the double mutant have been engineered in the context of the 929aa construct, and these proteins are currently being purified for future studies. This research will enable us to better understand how mutations in ERS contribute to disease etiology and may inform future therapeutic development.
Keywords: aminoacyl-tRNA synthetase