Poster abstracts

Poster number 96 submitted by Rylan Watkins

Investigating the Role of Trypanosoma brucei Prolyl-tRNA Synthetase and MCP3 in Maintaining tRNAPro Translational Fidelity

Rylan Watkins (Chemistry and Biochemistry, The Ohio State University), Irina Shulgina (Chemistry and Biochemistry, The Ohio State University), Juan D. Alfonzo (Microbiology, The Ohio State University ), Karin Musier-Forsyth (Chemistry and Biochemistry, The Ohio State University)

Trypanosoma brucei (Tb) are unicellular pathogens that cause Human African Trypanosomiasis (HAT)—a disease that is fatal if left untreated. Currently, HAT lacks a safe and effective treatment option; thus, new therapeutic targets encoded in the Tb genome are sought. Prolyl-tRNA synthetase (ProRS) attaches proline to cognate tRNAPro in a two-step aminoacylation reaction. ProRSs across all domains of life are error prone and often mischarge alanine onto cognate tRNAs. In most bacteria, ProRSs encode a dedicated editing domain (INS) that is responsible for hydrolyzing Ala-tRNAPro. Organisms that lack an INS domain within their ProRS often encode a homologous editing factor, ProXp-ala, to perform the same proofreading function in trans. Tb ProRS has an appended N-terminal ProXp-ala domain and belongs to a Multi-tRNA Synthetase Complex (MSC) along with an uncharacterized protein, MCP3, which has significant structural homology to ProXp-ala. MCP3 homologs and Tb-like ProRSs are highly conserved in human pathogens and are not encoded in the human genome. The Kinetoplastid Informatics Resource (TriTrypDB) reports MCP3 and ProRS are essential for parasite fitness. Here, we recombinantly expressed and purified Tb ProRS and MCP3 for the first time. We performed in vitro kinetic assays and showed that MCP3 has more robust Ala-tRNAPro editing activity than ProXp-ala enzymes from other organisms investigated to date. We performed site-directed mutagenesis and have identified several residues critical for efficient catalysis by MCP3. Multiple sequence alignments revealed that MCP3 has a unique N-terminal extension and we showed that this sequence is required for MCP3 dimerization. Preliminary kinetic data show that when expressed as a freestanding domain, Tb ProXp-ala displays reduced Ala-tRNAPro deacylase activity compared to full-length Tb ProRS. Prior to mammalian infection, trypanosomes rely on proline for ATP production. A consequence of this metabolic strategy is the excess side production of alanine. We hypothesize the exaggerated alanine pool in Tb results in more tRNAPro mischarging events and a need for two robust Ala-tRNAPro editing mechanisms. Our structural and mechanistic insights into tRNAPro editing in Tb serve as the foundation for exploring Tb ProRS and MCP3 as drug targets.

Keywords: aminoacyl-tRNA synthetase, translation fidelity, Multi-tRNA synthetase complex (MSC)