Post-transfer editing in vitro and in vivo by the β subunit of phenylalanyl-tRNA synthetase

Roy, Hervé; Ling, Jiqiang; Irnov, Irnov; Ibba, Michael

doi:10.1038/sj.emboj.7600474

Cited by 141 publications

(182 citation statements)

References 59 publications

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“…67. The hmt-PheRS variant S411 did not express well in regular LB media, suggesting that this residue might be important for maintaining protein stability or folding.…”

Section: Methodsmentioning

confidence: 99%

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Pathogenic mechanism of a human mitochondrial tRNA ^Phe mutation associated with myoclonic epilepsy with ragged red fibers syndrome

Ling

Roy

Qin

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Human mitochondrial tRNA (hmt-tRNA) mutations are associated with a variety of diseases including mitochondrial myopathies, diabetes, encephalopathies, and deafness. Because the current understanding of the precise molecular mechanisms of these mutations is limited, there is no efficient method to treat their associated mitochondrial diseases. Here, we use a variety of known mutations in hmt-tRNA Phe to investigate the mechanisms that lead to malfunctions. We tested the impact of hmt-tRNA Phe mutations on aminoacylation, structure, and translation elongation-factor binding. The majority of the mutants were pleiotropic, exhibiting defects in aminoacylation, global structure, and elongation-factor binding. One notable exception was the G34A anticodon mutation of hmt-tRNA Phe (mitochondrial DNA mutation G611A), which is associated with MERRF (myoclonic epilepsy with ragged red fibers). In vitro, the G34A mutation decreases aminoacylation activity by 100-fold, but does not affect global folding or recognition by elongation factor. Furthermore, G34A hmt-tRNA Phe does not undergo adenosine-to-inosine (A-to-I) editing, ruling out miscoding as a possible mechanism for mitochondrial malfunction. To improve the aminoacylation state of the mutant tRNA, we modified the tRNA binding domain of the nucleus-encoded human mitochondrial phenylalanyl-tRNA synthetase, which aminoacylates hmt-tRNA Phe with cognate phenylalanine. This variant enzyme displayed significantly improved aminoacylation efficiency for the G34A mutant, suggesting a general strategy to treat certain classes of mitochondrial diseases by modification of the corresponding nuclear gene.aminoacyl-tRNA synthetase ͉ translation ͉ mitochondria

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“…67. The hmt-PheRS variant S411 did not express well in regular LB media, suggesting that this residue might be important for maintaining protein stability or folding.…”

Section: Methodsmentioning

confidence: 99%

“…68; tRNAs transcripts and other His 6 -tagged proteins were purified as described in ref. 67. fMet-Phe dipeptide formation assay was performed as described in ref.…”

Section: Methodsmentioning

confidence: 99%

Pathogenic mechanism of a human mitochondrial tRNA ^Phe mutation associated with myoclonic epilepsy with ragged red fibers syndrome

Ling

Roy

Qin

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…MurM was solubilized prior to purification as described (21,38). S. pneumoniae tRNA Ala and tRNA Phe (wild type and U4G) and the E. coli wild type equivalents were produced by in vitro T7 RNA polymerase runoff transcription as described (22).…”

Section: Methodsmentioning

confidence: 99%

Lipid II-independent trans Editing of Mischarged tRNAs by the Penicillin Resistance Factor MurM

Shepherd¹,

Ibba

2013

Journal of Biological Chemistry

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Background: MurM utilizes aminoacyl-tRNAs and Lipid II for peptidoglycan biosynthesis in Streptococcus pneumoniae. Results: MurM deacylates mischarged aminoacyl-tRNAs in the absence of Lipid II. Conclusion:The ability of MurM to function in quality control can compensate for the absence of AlaXp proteins in S. pneumoniae. Significance: MurM can function in translation as a lipid-independent trans editing factor.

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“…Such mistranslation may lead to observable cellular and disease pathologies such as cell degeneration and apoptosis in mammalian systems (19) and neurodegeneration and ataxia in mice (20). Moreover, since heritable mutations in human glycyl-tRNA synthetase and tyrosyl-tRNA synthetase genes have been directly associated with the peripheral neuropathy Charcot-Marie-Tooth (21,22), the role of the aaRSs and their editing function in maintaining translational fidelity have become areas of intense study (23)(24)(25)(26)(27)(28).…”

mentioning

confidence: 99%

Quality control despite mistranslation caused by an ambiguous genetic code

Ruan

Palioura

Sabina

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

131

132

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A high level of accuracy during protein synthesis is considered essential for life. Aminoacyl-tRNA synthetases (aaRSs) translate the genetic code by ensuring the correct pairing of amino acids with their cognate tRNAs. Because some aaRSs also produce misacylated aminoacyl-tRNA (aa-tRNA) in vivo, we addressed the question of protein quality within the context of missense suppression by Cys-tRNA Pro , Ser-tRNA Thr , Glu-tRNA Gln , and Asp-tRNA Asn . Suppression of an active-site missense mutation leads to a mixture of inactive mutant protein (from translation with correctly acylated aa-tRNA) and active enzyme indistinguishable from the wild-type protein (from translation with misacylated aa-tRNA). Here, we provide genetic and biochemical evidence that under selective pressure, Escherichia coli not only tolerates the presence of misacylated aa-tRNA, but can even require it for growth. Furthermore, by using mass spectrometry of a reporter protein not subject to selection, we show that E. coli can survive the ambiguous genetic code imposed by misacylated aa-tRNA tolerating up to 10% of mismade protein. The editing function of aaRSs to hydrolyze misacylated aa-tRNA is not essential for survival, and the EF-Tu barrier against misacylated aa-tRNA is not absolute. Rather, E. coli copes with mistranslation by triggering the heat shock response that stimulates nonoptimized polypeptides to achieve a native conformation or to be degraded. In this way, E. coli ensures the presence of sufficient functional protein albeit at a considerable energetic cost.accuracy ͉ aminoacyl-tRNA ͉ fidelity ͉ protein synthesis ͉ missense suppression

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Post-transfer editing in vitro and in vivo by the β subunit of phenylalanyl-tRNA synthetase

Cited by 141 publications

References 59 publications

Pathogenic mechanism of a human mitochondrial tRNA ^Phe mutation associated with myoclonic epilepsy with ragged red fibers syndrome

Pathogenic mechanism of a human mitochondrial tRNA ^Phe mutation associated with myoclonic epilepsy with ragged red fibers syndrome

Lipid II-independent trans Editing of Mischarged tRNAs by the Penicillin Resistance Factor MurM

Quality control despite mistranslation caused by an ambiguous genetic code

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Post-transfer editing in vitro and in vivo by the β subunit of phenylalanyl-tRNA synthetase

Cited by 141 publications

References 59 publications

Pathogenic mechanism of a human mitochondrial tRNA Phe mutation associated with myoclonic epilepsy with ragged red fibers syndrome

Pathogenic mechanism of a human mitochondrial tRNA Phe mutation associated with myoclonic epilepsy with ragged red fibers syndrome

Lipid II-independent trans Editing of Mischarged tRNAs by the Penicillin Resistance Factor MurM

Quality control despite mistranslation caused by an ambiguous genetic code

Contact Info

Product

Resources

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Pathogenic mechanism of a human mitochondrial tRNA ^Phe mutation associated with myoclonic epilepsy with ragged red fibers syndrome

Pathogenic mechanism of a human mitochondrial tRNA ^Phe mutation associated with myoclonic epilepsy with ragged red fibers syndrome