Primary structure and functional expression of human Glycyl-tRNA synthetase, an autoantigen in myositis.

Gě, Qún; Trieu, Edward P.; Targoff, Ira N.

doi:10.1016/s0021-9258(19)61975-7

Cited by 43 publications

(8 citation statements)

References 52 publications

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“…They noted limited sequence similarity between histidyl-tRNA synthetase and several other aminoacyl-tRNA synthetases (including glycyl-tRNA synthetase) and the E. coli seryl-tRNA synthetase over a 32amino acid residue region. The similarity of the N-terminal motifs in B. mori and human (Ge et a!., 1994;Shiba et al, 1994;Williams et al, 1995) glycyl-tRNA synthetase to the corresponding sequence in the human histidine enzyme and conformational studies of synthetic peptides based on the sequence of the histidine enzyme suggest that the N-terminal structure forms an antiparallel coiled coil, similar to the N-terminus of E. coli seryl-tRNA synthetase (Cusack et al, 1990). The absence of detectable effects of its removal on the Michaelis constant for tRNA may indicate that the Michaelis constant does not reflect binding but is a complex kinetic constant.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Truncated Forms of Bombyx mori Glycyl-tRNA Synthetase: Function of an N-Terminal Structure in RNA Binding

Nada²,

Dignam³

1995

Biochemistry

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Bombyx mori glycyl-tRNA synthetase (GRS) was expressed as the full length protein and as N-terminally and C-terminally truncated forms. The intact enzyme and forms with deletions of 12, 27, 46, and 55 N-terminal residues were expressed, purified, and characterized. All were active, having 15-25% of both pyrophosphate exchange activity and aminoacyl-tRNA synthetase activity compared to wild type enzyme. Active site titration indicated that this difference in activity was not the result of production of inactive enzyme. Sedimentation and gel filtration experiments indicate that the N-terminally deleted forms and the wild type enzyme were dimers. Deletion of 55 N-terminal residues did not result in significant effects on the Michaelis constants for ATP, glycine, or tRNA, while deletion of 108 N-terminal residues and two internal 64- and 200-residue deletions generated inactive forms. Five forms with C-terminal deletions of 24, 37, 59, 162, and 327 amino acid residues were soluble and intact but lacked detectable pyrophosphate exchange activity or aminoacyl-tRNA synthetase activity. The C-terminal sequence may be required for catalysis or to maintain a stable structure. Zone electrophoresis demonstrated the wild type enzyme bound both tRNA(Gly) and noncognate tRNA(Ala). Deletion of 55 N-terminal residues resulted in altered binding of tRNA(Gly) and eliminated binding of tRNA(Ala). The first 55 N-terminal residues are not essential for catalysis, dimerization, or substrate binding in aminoacylation but are required for RNA binding not associated with aminoacylation.

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Section: Discussionmentioning

confidence: 99%

Analysis of Truncated Forms of Bombyx mori Glycyl-tRNA Synthetase: Function of an N-Terminal Structure in RNA Binding

Nada²,

Dignam³

1995

Biochemistry

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“…Purified protein was lyophilized and stored at -20 °C. A uniformly 15 N-labeled protein was prepared from cells grown in M9 minimal medium containing 1.0 g of 15 NH 4 Cl/L and 2.0 g of glucose/L, and glucose was substituted by 13 C-glucose for the simultaneous isotope labeling of 13 C and 15 N. NMR samples were prepared by dissolving about 2 mM protein in 0.5 mL of either 90% H 2 O/10% 2 H 2 O or 99.9% 2 H 2 O, and the pH was adjusted to 5.0 ( 0.05 (glass electrode, uncorrected) with concentrated NaO 2 H. Purified protein was verified by N-terminal amino acid sequence at Korea Basic Science Institute (KBSI) in Seoul. The expressed protein of EPRS-R1 was cleaved for 10 min at 0 °C with subtilisin instead of thrombin to remove other flanking residues in addition to the histidine-tag.…”

Section: Methodsmentioning

confidence: 99%

Structural Analysis of Multifunctional Peptide Motifs in Human Bifunctional tRNA Synthetase: Identification of RNA-Binding Residues and Functional Implications for Tandem Repeats^,

et al. 2000

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Human bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) contains three tandem repeats linking the two catalytic domains. These repeated motifs have been shown to be involved in protein-protein and protein-nucleic acid interactions. The single copy of the homologous motifs has also been found in several different aminoacyl-tRNA synthetases. The solution structure of repeat 1 (EPRS-R1) and the secondary structure of the whole appended domain containing three repeated motifs in EPRS (EPRS-R123) was determined by nuclear magnetic resonance (NMR) spectroscopy. EPRS-R1 consists of two helices (residues 679-699 and 702-721) arranged in a helix-turn-helix, which is similar to other RNA binding proteins and the j-domain of DnaJ, and EPRS-R123 is composed of three helix-turn-helix motifs linked by an unstructured loop. When tRNA is bound to the appended domain, chemical shifts of several residues in each repeat are perturbed. However, the perturbed residues in each repeat are not the same although they are in the same binding surface, suggesting that each repeat in the appended domain is dynamically arranged to maximize contacts with tRNA. The affinity of tRNA to the three-repeated motif was much higher than to the single motif. These results indicate that each of the repeated motifs has a weak intrinsic affinity for tRNA, but the repetition of the motifs may be required to enhance binding affinity. Thus, the results of this work gave information on the RNA-binding mode of the multifunctional peptide motif attached to different ARSs and the functional reason for the repetition of this motif.

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“…Like human TyrRS, human TrpRS contains an appended domain that is found only in mammalian TrpRSs (Figure 1). In this instance, the extra domain is appended to the N-terminus and is similar to a domain found in eukaryote tRNA synthetases for histidine, glycine, and methionine (35)(36)(37). It is also repeated as three tandem domains that join human glutamyl-with prolyl-tRNA synthetase to give the Glu-Pro tRNA synthetase that is found in higher eukaryotes (38).…”

Section: Translation Proteins and Cell Signaling: A New Paradigm Emap...mentioning

confidence: 99%

Activation of Angiogenic Signaling Pathways by Two Human tRNA Synthetases

Ewalt

Schimmel

2002

Biochemistry

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Aminoacyl-tRNA synthetases establish the rules of the genetic code by joining amino acids to tRNAs that bear the anticodon triplets corresponding to the attached amino acids. The enzymes are thought to be among the earliest proteins to appear, in the transition from a putative RNA world to the theater of proteins. Over their long evolution, the enzymes have acquired additional functions that typically require specialized insertions or domain fusions. Recently, fragments of the closely related human tyrosyl- and tryptophanyl-tRNA synthetases were discovered to be active in angiogenesis signaling pathways. One synthetase fragment has proangiogenic activity, while the other is antiangiogenic. Activity was demonstrated in cell-based assays in vitro and in vivo in the chick embryo, and in the neonatal and adult mouse. The full-length, native enzymes are inactive in these same assays. Activation of angiogenesis activity requires fragment production from the native enzymes by protease cleavage or by translation of alternatively spliced pre-mRNA. Thus, these tRNA synthetases link translation to a major cell-signaling pathway in mammalian cells. The results with animals suggest that therapeutic applications are possible with these tRNA synthetases.

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Primary structure and functional expression of human Glycyl-tRNA synthetase, an autoantigen in myositis.

Cited by 43 publications

References 52 publications

Analysis of Truncated Forms of Bombyx mori Glycyl-tRNA Synthetase: Function of an N-Terminal Structure in RNA Binding

Analysis of Truncated Forms of Bombyx mori Glycyl-tRNA Synthetase: Function of an N-Terminal Structure in RNA Binding

Structural Analysis of Multifunctional Peptide Motifs in Human Bifunctional tRNA Synthetase: Identification of RNA-Binding Residues and Functional Implications for Tandem Repeats^,

Activation of Angiogenic Signaling Pathways by Two Human tRNA Synthetases

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Primary structure and functional expression of human Glycyl-tRNA synthetase, an autoantigen in myositis.

Cited by 43 publications

References 52 publications

Analysis of Truncated Forms of Bombyx mori Glycyl-tRNA Synthetase: Function of an N-Terminal Structure in RNA Binding

Analysis of Truncated Forms of Bombyx mori Glycyl-tRNA Synthetase: Function of an N-Terminal Structure in RNA Binding

Structural Analysis of Multifunctional Peptide Motifs in Human Bifunctional tRNA Synthetase: Identification of RNA-Binding Residues and Functional Implications for Tandem Repeats,

Activation of Angiogenic Signaling Pathways by Two Human tRNA Synthetases

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Structural Analysis of Multifunctional Peptide Motifs in Human Bifunctional tRNA Synthetase: Identification of RNA-Binding Residues and Functional Implications for Tandem Repeats^,