Expression and characterization of a human mitochondrial phenylalanyl-tRNA synthetase 1 1Edited by D. E. Draper

Bullard, James M.; Cai, Ying; Demeler, Borries; Spremulli, Linda L.

doi:10.1006/jmbi.1999.2708

Cited by 74 publications

(79 citation statements)

References 26 publications

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“…The D400N substitution also decreased recognition of the WT hmt-tRNA Phe by 3-fold, indicating specific interactions between residue 400 of hmt-PheRS and position 34 of hmt-tRNA Phe . This is also consistent with previous predictions that the C-terminal domain of hmt-PheRS is involved in anticodon recognition (58). In an attempt to further increase the aminoacylation activity of the G34A mutant, we introduced the additional replacements S411 or R450 into hmt-PheRS D400N (Fig.…”

Section: Rescue Of G34a Hmt-trna Phe Aminoacylation Defect By Hmt-pherssupporting

confidence: 88%

“…In an attempt to explore potential routes for gene therapy, we modified the tRNA binding region of nuclear-encoded human mitochondrial PheRS. The resulting enzyme variants showed partially restored aminoacylation efficiency for G34A hmt-tRNA Phe and confirmed predictions that the C terminus of hmt-PheRS is involved in anticodon binding (58,66). Because the G34A mutation of hmt-tRNA Phe does not affect other aspects of tRNA function, a modified nuclear gene for hmt-PheRS may be able to restore the function of mitochondria containing this mutation via allotopic expression.…”

Section: Restoring the Function Of A Mutant Trna That Causes Myoclonicsupporting

confidence: 66%

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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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Section: Rescue Of G34a Hmt-trna Phe Aminoacylation Defect By Hmt-pherssupporting

confidence: 88%

Section: Restoring the Function Of A Mutant Trna That Causes Myoclonicsupporting

confidence: 66%

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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“…This seems to be accompanied by a process of size reduction in mitochondrial ARS (20,21) and a dramatic divergence in the structure of tRNAs (22). The elimination of tRNA genes in Kinetoplastida mitochondria may be seen as another example of integration of the cell's genetic codes.…”

Section: Discussionmentioning

confidence: 99%

A mechanism for functional segregation of mitochondrial and cytosolic genetic codes

Español

Thut

Schneider

et al. 2009

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

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The coexistence of multiple gene translation machineries is a feature of eukaryotic cells and a result of the endosymbiotic events that gave rise to mitochondria, plastids, and other organelles. The conditions required for the integration of these apparatuses within a single cell are not understood, but current evidence indicates that complete ablation of the mitochondrial protein synthesis apparatus and its substitution by its cytosolic equivalent is not possible. Why certain mitochondrial components and not others can be substituted by cytosolic equivalents is not known. In trypanosomatids this situation reaches a limit, because certain aminoacyl-tRNA synthetases are mitochondrial specific despite the fact that all tRNAs in these organisms are shared between cytosol and mitochondria. Here we report that a mitochondria-specific lysyl-tRNA synthetase in Trypanosoma has evolved a mechanism to block the activity of the enzyme during its synthesis and translocation. Only when the enzyme reaches the mitochondria is it activated through the cleavage of a C-terminal structural extension, preventing the possibility of the enzyme being active in the cytosol.aminoacyl-tRNA synthetases ͉ mitochondria ͉ tRNA ͉ trypanosoma

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“…In order to test these two possibilities, we attempted to measure binding constants for tRNA Phe using fluorescence techniques. It has been estimated previously that mtPheRS has a K m for tRNA Phe of $18 lM [15] which, given the low yields of active mitochondrial tRNA Phe transcribed in vitro, precludes accurate determination of binding constants. Comparison of binding at sub-saturating non-reducing concentrations suggests that WT mtPheRS binds tRNA Phe better than the closed mutant form (Fig.…”

Section: Trna Binding By Mtphersmentioning

confidence: 99%

“…PheRS is instead active as a monomer in the organelles of eukaryotes [14,15] and lacks an editing function [16]. Human mitochondrial PheRS (mtPheRS) is highly homologous to the corresponding domains of bacterial PheRS, consisting of an N-terminal catalytic a domain fused to a C-terminal B8-like anticodon-binding domain (ABD) from the b subunit [17].…”

Section: Introductionmentioning

confidence: 99%

Large‐scale movement of functional domains facilitates aminoacylation by human mitochondrial phenylalanyl‐tRNA synthetase

et al. 2009

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a b s t r a c tStructural studies suggest rearrangement of the RNA-binding and catalytic domains of human mitochondrial PheRS (mtPheRS) is required for aminoacylation. Crosslinking the catalytic and RNA-binding domains resulted in a ''closed" form of mtPheRS that still catalyzed ATP-dependent Phe activation, but was no longer able to transfer Phe to tRNA and complete the aminoacylation reaction. SAXS experiments indicated the presence of both the closed and open forms of mtPheRS in solution. Together, these results indicate that conformational flexibility of the two functional modules in mtPheRS is essential for its phenylalanylation activity. This is consistent with the evolution of the aminoacyl-tRNA synthetases as modular enzymes consisting of separate domains that display independent activities.

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Expression and characterization of a human mitochondrial phenylalanyl-tRNA synthetase 1 1Edited by D. E. Draper

Cited by 74 publications

References 26 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

A mechanism for functional segregation of mitochondrial and cytosolic genetic codes

Large‐scale movement of functional domains facilitates aminoacylation by human mitochondrial phenylalanyl‐tRNA synthetase

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Expression and characterization of a human mitochondrial phenylalanyl-tRNA synthetase 1 1Edited by D. E. Draper

Cited by 74 publications

References 26 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

A mechanism for functional segregation of mitochondrial and cytosolic genetic codes

Large‐scale movement of functional domains facilitates aminoacylation by human mitochondrial phenylalanyl‐tRNA synthetase

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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