Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

Ling, Jiqiang; Peterson, K.M.; Simonović, Ivana; Cho, Chris; Söll, Dieter; Simonović, Miljan

doi:10.1073/pnas.1200109109

Cited by 27 publications

(46 citation statements)

References 39 publications

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“…Enzyme concentrations above 0.25 M led to a loss in the linear dependence on enzyme activity; thus, 0.25 M enzyme was used in subsequent studies. The specific activity of the enzyme under these conditions was 1.4 min Ϫ1 , which is comparable to published values for the yeast enzyme (k cat ϭ 2.8 min Ϫ1 [52]). To provide chemical validation of ThrRS as a drug target in T. brucei, we tested the reported ThrRS inhibitor borrelidin against both the T. brucei ThrRS enzyme and, for activity, the BSF T. brucei cells in vitro.…”

Section: Gene Selectionsupporting

confidence: 86%

Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

et al. 2014

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bHuman African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyltRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (␣ and ␤) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts.

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Section: Gene Selectionsupporting

confidence: 86%

Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

et al. 2014

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“…The overall structure of the binary complex is similar to the previously reported structure of MST1 complexed with the nonhydrolyzable analog of Thr-AMP (TAM) (36), with the main differences noted in the conformations of the active-site lid and the anticodon-binding domain. The binary complex crystals, which belonged to a monoclinic space group (C2 1 ), contained two MST1 homodimers in the asymmetric unit.…”

Section: Binding Of the Ser-amp Analog Stabilizes The Active-site Lidsupporting

confidence: 78%

“…2 in Ref. 36), the interactions of the seryl moiety with Zn 2ϩ do not fully resemble that of the threonyl moiety. In particular, the ␥-OH group of SAM is positioned 2.4 and 3.1 Å away from the O␦1 atom of Asp182 and Zn 2ϩ , respectively, whereas the same distances in the MST1-TAM binary complex crystal were 2.6 and 2.2 Å (data not shown).…”

Section: Differences In the Binding Of Ser-amp And Thr-amp To The Amimentioning

confidence: 91%

“…Yeast MST1 is homologous to bacterial ThrRSs but lacks the N-terminal editing domain that hydrolyzes misacylated Ser-tRNA Thr (35,36), prompting us to investigate the fidelity of MST1 for different nearcognate amino acids such as Ser, Val, Ala, and Cys. We first measured the activation rates of Thr and Ser by MST1 using a pyrophosphate exchange assay.…”

Section: Mst1 Misactivates and Edits Serine In The Absence Ofmentioning

confidence: 99%

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The Mechanism of Pre-transfer Editing in Yeast Mitochondrial Threonyl-tRNA Synthetase

Ling¹,

Peterson

Simonović

et al. 2012

Journal of Biological Chemistry

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Background:The mechanism of pre-transfer editing by which aaRSs regulate translational fidelity is not well understood. Results: Yeast mitochondrial ThrRS, MST1, hydrolyzes seryl adenylate at the aminoacylation active site more rapidly than the cognate threonyl adenylate. Conclusion: MST1 discriminates against serine and reduces mischarging of threonine tRNA by employing pre-transfer editing. Significance: The mechanism of misactivation and pre-transfer editing of serine by ThrRS is provided.

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“…Bacterial and eukaryotic cytoplasmic ThrRS, shown to misactivate non-cognate Ser, mainly use an efficient post-transfer editing activity to clear mischarged Ser-tRNA Thr in addition to tRNAdependent pretransfer editing (18 -20). In contrast, Saccharomyces cerevisiae mitochondrial ThrRS (ScmtThrRS) naturally lacks an editing domain and only harbors tRNA-independent and tRNA isoacceptor-specific pretransfer editing (21,22). As a result, ScmtThrRS lacks post-transfer editing and is an errorprone synthetase, forming Ser-tRNA Thr , at least in vitro (22).…”

mentioning

confidence: 99%

A Human Disease-causing Point Mutation in Mitochondrial Threonyl-tRNA Synthetase Induces Both Structural and Functional Defects

Wang

Zhou

Ruan

et al. 2016

Journal of Biological Chemistry

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Mitochondria require all translational components, including aminoacyl-tRNA synthetases (aaRSs), to complete organelle protein synthesis. Some aaRS mutations cause mitochondrial disorders, including human mitochondrial threonyl-tRNA synthetase (hmtThrRS) (encoded by TARS2), the P282L mutation of which causes mitochondrial encephalomyopathies. However, its catalytic and structural consequences remain unclear. Herein, we cloned TARS2 and purified the wild-type and P282L mutant hmtThrRS. hmtThrRS misactivates non-cognate Ser and uses post-transfer editing to clear erroneously synthesized products. In vitro and in vivo analyses revealed that the mutation induces a decrease in Thr activation, aminoacylation, and proofreading activities and a change in the protein structure and/or stability, which might cause reduced catalytic efficiency. We also identified a splicing variant of TARS2 mRNA lacking exons 8 and 9, the protein product of which is targeted into mitochondria. In HEK293T cells, the variant does not dimerize and cannot complement the ThrRS knock-out strain in yeast, suggesting that the truncated protein is inactive and might have a noncanonical function, as observed for other aaRS fragments. The present study describes the aminoacylation and editing properties of hmtThrRS, clarifies the molecular consequences of the P282L mutation, and shows that the yeast ThrRS-deletion model is suitable to test pathology-associated point mutations or alternative splicing variants of mammalian aaRS mRNAs.Aminoacyl-tRNA synthetases (aaRSs) 4 supply the ribosome with aminoacyl-tRNA substrates for protein synthesis (1, 2). This process starts with amino acid activation by condensation with ATP to form the aminoacyl adenylate aa-AMP and pyrophosphate. The activated amino acid then reacts with the 3Ј-end of the cognate tRNA to yield the aminoacyl-tRNA (aatRNA), which is transferred by EF-Tu to the protein biosynthesis machinery as a building block. Highly accurate protein synthesis is indispensable for cell growth. To maintain fidelity during protein synthesis, aaRS needs to select the correct amino acid and tRNA substrates from a large number of structurally similar molecules in the cells. The specificity of aaRS is greatly challenged by the presence of the 20 amino acids in which the physicochemical properties are sometimes very similar and by non-proteogenous amino acids and diverse metabolites produced by cell metabolism. aaRSs that do not show an overall selectivity above 1 in 3000 are predicted to require proofreading (editing) activity to maintain sufficient accuracy during aa-tRNA synthesis (3). In fact, editing activity has evolved in half of the currently identified aaRS to remove aberrantly produced aa-AMP (pretransfer editing) and/or aa-tRNA (posttransfer editing) (4). tRNA mischarging may be toxic and deleterious for cells. Even a slight decrease in aminoacylation accuracy could cause an intracellular accumulation of misfolded proteins and up-regulation of cytoplasmic protein chaperones in neurons, leading to severe...

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Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

Cited by 27 publications

References 39 publications

Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

The Mechanism of Pre-transfer Editing in Yeast Mitochondrial Threonyl-tRNA Synthetase

A Human Disease-causing Point Mutation in Mitochondrial Threonyl-tRNA Synthetase Induces Both Structural and Functional Defects

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