Discriminator Base is a Critical Recognition Element for Trypanosoma brucei Ala-tRNAPro Editing Domains
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Date
2023-05
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The Ohio State University
Abstract
Prolyl-tRNA synthetases (ProRSs) mischarge Ala onto cognate tRNAPro. In bacteria, this error can be corrected by a cis-editing domain appended to the synthetase (INS) or by a homologous freestanding trans-editing domain, ProXp-ala. Trypanosoma brucei (Tb) ProRS encodes an appended ProXp-ala domain and a unique free-standing INS homolog—MCP3 (multi-tRNA synthetase complex 3 protein). We hypothesize that Tb may have two Ala-tRNAPro editing domains due to its unusual metabolism. In the procylic form, the main carbon source is proline, with alanine produced as a metabolic by-product. With high intracellular concentrations of alanine, robust editing mechanisms may be needed to avoid misincorporation of Ala at Pro codons. Free-standing Homo sapiens (Hs) ProXp-ala recognizes nucleotides in the tRNA acceptor stem for optimal function. In contrast, the ProRS-appended E. coli INS domain relies exclusively on ProRS anticodon recognition. Here, we probed the acceptor stem specificity of Tb Ala-tRNAPro editing domains. Ala-tRNAPro deacylation assays revealed that the activities of ProRS-appended Tb ProXp-ala, as well as free-standing ProXp-ala and MCP3, are significantly decreased upon mutation of the discriminator base (C73A), with more relaxed recognition of the first base pair (G1:C72). Surprisingly, MCP3 deacylated the triple mutant (G1C:C72G, C73A) Ala-tRNAPro better than the C73A single mutant, and displayed robust deacylation of G1:C72, A73-containing Ala-tRNAAla in vitro. Thus, the context of the discriminator base and the Ala moiety appear to be important determinants for this editing domain. As MCP3 is not encoded in the human genome, results of these studies may have implications for new therapeutic strategies.
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Keywords
Aminoacyl-tRNA synthetase, tRNA, Trypanosoma brucei, Microbiology, Translational fidelity