Purification and in vitro characterization of Trypanosoma brucei prolyl-tRNA synthetase
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Date
2022-05
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The Ohio State University
Abstract
Housekeeping proteins include a broad variety of enzymes that complete the basic tasks
necessary for cell survival. Among this group, aminoacyl-tRNA synthetases (aaRSs) function to
attach the correct amino acid to the cognate tRNA substrate in a reaction known as
aminoacylation. The aminoacyl-tRNA is delivered to the ribosome by an elongation factor,
where it participates in protein synthesis. While catalysis of the aminoacylation reaction seems
straightforward, aaRSs must sample similarly sized amino acids to find a match for the protein
active site. Amino acids with the same or smaller molecular volume can fit into the active site
and become mistakenly charged to noncognate tRNA. Thus, the cell uses proofreading
mechanisms to ensure that only cognate aminoacyl-tRNAs arrive at the ribosome. In the context
of charging L-proline to tRNAPro, prolyl-tRNA synthetase (ProRS) frequently mischarges
alanine and cysteine and generates Ala-tRNAPro and Cys-tRNA
Pro. The bacterial Escherichia coli
enzyme, for example, relies on an insertion (INS) domain in its ProRS architecture to hydrolyze
mischarged Ala-tRNAPro species. Other prokaryotes such as Caulobacter crescentus, lack this
embedded domain but encode a homologous, free-standing editing factor, ProXp-ala, for the
same function. Beyond prokarya, however, understanding of eukaryotic editing processes
remains limited. Homo sapiens and other higher eukaryotes also possess a free-standing ProXp ala domain for editing. Bioinformatic analyses have revealed lower eukaryotes with a ProRS
architecture reminiscent of E. coli. These eukaryotes encode a canonical ProRS with a fused N terminal ProXp-ala domain. Because many organisms with this understudied ProRS structure are
parasites such as Plasmodium falciparum, Leishmania tarentolae, and Trypanosoma brucei,
potential nuances in tRNA recognition and editing mechanism hold promise for drug targeting.
Inhibition of Trypanosoma brucei (Tb) ProRS editing, for example, may provide a cure for
African Sleeping Sickness while avoiding the human system entirely. In this work, we attempted
to overexpress and purify full-length Tb ProRS for the first time in E. coli. While purification of
the full-length construct was challenging due to poor solubility and low yields, the individual
ProXp-ala and ΔProXp-Ala ProRS constructs were generated via SLIM-PCR, expressed, and
purified with appreciable yield. The Tb ΔProXp-Ala ProRS enzyme activity was probed via
aminoacylation reactions. Pure Tb tRNAPro was generated by in vitro transcription and used in
aminoacylation reactions with the Tb ProRS domain to generate Pro-tRNAPro. Flexizyme
charging was used to generate Tb Ala-tRNAPro. Deacylation assays were performed with the
purified Tb ProXp-ala domain to provide preliminary kinetic data demonstrating the editing
capabilities of Tb ProRS. Although these assays provide no explanation for the selective pressure
driving this fused architecture, they do provide preliminary evidence that Tb ProXp-ala is an
Ala-tRNAPro deacylase in vitro. Comparison to other trans editing factors revealed that the
isolated Tb ProXp-ala domain is a weaker deacylase; however, the presence of the fused ProRS
domain may be required for more robust deacylation activity and future work will address this
hypothesis. Overall, we have demonstrated that the isolated Tb ProRS domains—both behave as
predicted by bioinformatic analysis and provide further impetus for the purification of the full-length protein.
Description
Keywords
Trypanosomiasis, aminoacyl-tRNA synthetase, transfer RNA, deacylation, Amino acids