scholarly journals Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand

1977 ◽  
Vol 167 (2) ◽  
pp. 419-428 ◽  
Author(s):  
C M Clarke ◽  
J R Knowles
Keyword(s):  
Ribonucleic Acid ◽  
Bacillus Stearothermophilus ◽  
Preceding Paper ◽  
Trna Synthetase ◽  
Rapid Preparation ◽  
Purification Method ◽  
Trna Synthetases ◽  
Trna Species ◽  
Aminoacyl Transfer ◽  
Cognate Trna

The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417].

scholarly journals Adenosine triphosphate consumption by bacterial arginyl-transfer ribonucleic acid synthetases

1979 ◽  
Vol 179 (2) ◽  
pp. 407-412 ◽  
Author(s):  
J M Godeau ◽  
J Charlier
Keyword(s):  
Escherichia Coli ◽  
Adenosine Triphosphate ◽  
Ribonucleic Acid ◽  
Adenosine Triphosphatase ◽  
Bacillus Stearothermophilus ◽  
Trna Synthetase ◽  
Luciferase Assay ◽  
Trna Synthetases ◽  
E Coli ◽  
Firefly Luciferin

ATP consumption by arginyl-tRNA synthetases from Escherichia coli and Bacillus stearothermophilus has been investigated by the firefly luciferin–luciferase assay. Arginyl-tRNA synthetase from E. coli utilizes ATP only for aminocylation of tRNA with a 1:1 stoicheiometry. In contrast, we have shown an adenosine triphosphatase activity of arginyl-tRNA synthetase from B. stearothermophilus in the absence of tRNAArg. Dowex chromatography revealed the formation of ADP by the thermophile enzyme; under aminoacylation conditions, AMP was also formed in amounts stoicheiometric with arginyl-tRNA formation.

scholarly journals The Evolutionary Fate of Mitochondrial Aminoacyl-tRNA Synthetases in Amitochondrial Organisms

2021 ◽  
Author(s):  
Gabor L. Igloi
Keyword(s):  
Protein Sequence ◽  
Identity Element ◽  
Mitochondrial Gene ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Cognate Trna ◽  
Evolutionary Fate ◽  
Endosymbiotic Evolution

AbstractDuring the endosymbiotic evolution of mitochondria, the genes for aminoacyl-tRNA synthetases were transferred to the ancestral nucleus. A further reduction of mitochondrial function resulted in mitochondrion-related organisms (MRO) with a loss of the organelle genome. The fate of the now redundant ancestral mitochondrial aminoacyl-tRNA synthetase genes is uncertain. The derived protein sequence for arginyl-tRNA synthetase from thirty mitosomal organisms have been classified as originating from the ancestral nuclear or mitochondrial gene and compared to the identity element at position 20 of the cognate tRNA that distinguishes the two enzyme forms. The evolutionary choice between loss and retention of the ancestral mitochondrial gene for arginyl-tRNA synthetase reflects the coevolution of arginyl-tRNA synthetase and tRNA identity elements.

scholarly journals Cross-editing by a tRNA synthetase allows vertebrates to abundantly express mischargeable tRNA without causing mistranslation

2020 ◽  
Vol 48 (12) ◽  
pp. 6445-6457 ◽  
Author(s):  
Meirong Chen ◽  
Bernhard Kuhle ◽  
Jolene Diedrich ◽  
Ze Liu ◽  
James J Moresco ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Trna Synthetase ◽  
Intrinsic Activity ◽  
Trna Synthetases ◽  
Evolutionarily Conserved ◽  
Trna Species ◽  
In Trans ◽  
Editing Activity

Abstract The accuracy in pairing tRNAs with correct amino acids by aminoacyl-tRNA synthetases (aaRSs) dictates the fidelity of translation. To ensure fidelity, multiple aaRSs developed editing functions that remove a wrong amino acid from tRNA before it reaches the ribosome. However, no specific mechanism within an aaRS is known to handle the scenario where a cognate amino acid is mischarged onto a wrong tRNA, as exemplified by AlaRS mischarging alanine to G4:U69-containing tRNAThr. Here, we report that the mischargeable G4:U69-containing tRNAThr are strictly conserved in vertebrates and are ubiquitously and abundantly expressed in mammalian cells and tissues. Although these tRNAs are efficiently mischarged, no corresponding Thr-to-Ala mistranslation is detectable. Mistranslation is prevented by a robust proofreading activity of ThrRS towards Ala-tRNAThr. Therefore, while wrong amino acids are corrected within an aaRS, a wrong tRNA is handled in trans by an aaRS cognate to the mischarged tRNA species. Interestingly, although Ala-tRNAThr mischarging is not known to occur in bacteria, Escherichia coli ThrRS also possesses robust cross-editing ability. We propose that the cross-editing activity of ThrRS is evolutionarily conserved and that this intrinsic activity allows G4:U69-containing tRNAThr to emerge and be preserved in vertebrates to have alternative functions without compromising translational fidelity.

scholarly journals Universal pathway for posttransfer editing reactions: Insights from the crystal structure of TtPheRS with puromycin

2015 ◽  
Vol 112 (13) ◽  
pp. 3967-3972 ◽  
Author(s):  
Dmitry Tworowski ◽  
Liron Klipcan ◽  
Moshe Peretz ◽  
Nina Moor ◽  
Mark G. Safro
Keyword(s):  
Crystal Structure ◽  
Thermus Thermophilus ◽  
Complex Structure ◽  
Trna Synthetase ◽  
Editing Site ◽  
Mutual Transformation ◽  
Trna Synthetases ◽  
Trna Species ◽  
Amino Acid Binding ◽  
Cis And Trans

At the amino acid binding and recognition step, phenylalanyl-tRNA synthetase (PheRS) faces the challenge of discrimination between cognate phenylalanine and closely similar noncognate tyrosine. Resampling of Tyr-tRNAPhe to PheRS increasing the number of correctly charged tRNA molecules has recently been revealed. Thus, the very same editing site of PheRS promotes hydrolysis of misacylated tRNA species, associated both with cis- and trans-editing pathways. Here we report the crystal structure of Thermus thermophilus PheRS (TtPheRS) at 2.6 Å resolution, in complex with phenylalanine and antibiotic puromycin mimicking the A76 of tRNA acylated with tyrosine. Starting from the complex structure and using a hybrid quantum mechanics/molecular mechanics approach, we investigate the pathways of editing reaction catalyzed by TtPheRS. We show that both 2′ and 3′ isomeric esters undergo mutual transformation via the cyclic intermediate orthoester, and the editing site can readily accommodate a model of Tyr-tRNAPhe where deacylation occurs from either the 2′- or 3′-OH. The suggested pathway of the hydrolytic reaction at the editing site of PheRS is of sufficient generality to warrant comparison with other class I and class II aminoacyl-tRNA synthetases.

scholarly journals Myocardial aminoacyl-transfer-ribonucleic acid synthetase and aminoacyl-transferring enzyme activity

1972 ◽  
Vol 126 (2) ◽  
pp. 409-416 ◽  
Author(s):  
K. Gibson ◽  
P. Harris
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Ribonucleic Acid ◽  
Trna Synthetase ◽  
Transferase Activity ◽  
Synthetase Activity ◽  
Aminoacyl Trna Synthetase ◽  
Standard Preparation ◽  
Enzyme Systems ◽  
Aminoacyl Transfer

The properties of cytoplasmic aminoacyl-tRNA synthetase and aminoacyl-transferring enzymes in the myocardium were examined and methods for the assay of the activity of these enzyme systems were developed. Aminoacyl-tRNA synthetase activity was measured from the rate of incorporation of 14C-labelled amino acid into aminoacyl-tRNA. Transferase activity was measured from the rate of incorporation of amino[14C]acyl-tRNA into protein in the presence of a standard preparation of hepatic ribosomes. Aminoacyl-tRNA synthetase activity is labile once the heart has been homogenized, whereas transferase activity is stable. The source of energy for synthetase activity is ATP; that for transferase is GTP. Transferase activity was inhibited by puromycin and stimulated by dithiothreitol, whereas synthetase activity was unaffected.

scholarly journals Fine-Tuning of Alanyl-tRNA Synthetase Quality Control Alleviates Global Dysregulation of the Proteome

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1222
Author(s):  
Paul Kelly ◽  
Arundhati Kavoor ◽  
Michael Ibba
Keyword(s):  
Amino Acids ◽  
Quality Control ◽  
Amino Acid ◽  
Antibiotic Sensitivity ◽  
Trna Synthetase ◽  
Fine Tuning ◽  
Trna Synthetases ◽  
E Coli ◽  
Suppressor Mutations ◽  
Cognate Trna

One integral step in the transition from a nucleic acid encoded-genome to functional proteins is the aminoacylation of tRNA molecules. To perform this activity, aminoacyl-tRNA synthetases (aaRSs) activate free amino acids in the cell forming an aminoacyl-adenylate before transferring the amino acid on to its cognate tRNA. These newly formed aminoacyl-tRNA (aa-tRNA) can then be used by the ribosome during mRNA decoding. In Escherichia coli, there are twenty aaRSs encoded in the genome, each of which corresponds to one of the twenty proteinogenic amino acids used in translation. Given the shared chemicophysical properties of many amino acids, aaRSs have evolved mechanisms to prevent erroneous aa-tRNA formation with non-cognate amino acid substrates. Of particular interest is the post-transfer proofreading activity of alanyl-tRNA synthetase (AlaRS) which prevents the accumulation of Ser-tRNAAla and Gly-tRNAAla in the cell. We have previously shown that defects in AlaRS proofreading of Ser-tRNAAla lead to global dysregulation of the E. coli proteome, subsequently causing defects in growth, motility, and antibiotic sensitivity. Here we report second-site AlaRS suppressor mutations that alleviate the aforementioned phenotypes, revealing previously uncharacterized residues within the AlaRS proofreading domain that function in quality control.

scholarly journals Probing the substrate-binding sites of aminoacyl-tRNA synthetases with the procion dye green HE-4BD

1989 ◽  
Vol 258 (3) ◽  
pp. 715-721 ◽  
Author(s):  
J E C McArdell ◽  
M Duffield ◽  
T Atkinson
Keyword(s):  
Bacillus Stearothermophilus ◽  
Competitive Inhibitor ◽  
Reactive Dye ◽  
Trna Synthetase ◽  
Enzyme Inactivation ◽  
Trna Synthetases ◽  
Time Period ◽  
Substrate Binding Sites ◽  
Methionyl Trna Synthetase ◽  
Fold Excess

A reactive bis-dichloro derivative of the Procion dye Green HE-4BD was shown to inactivate irreversibly methionyl-tRNA synthetase (MTS) from Escherichia coli and also tryptophyl-tRNA synthetase (WTS) and tyrosyl-tRNA synthetase (YTS) from Bacillus stearothermophilus at pH 8.5 and 37 degrees C. At a 5-fold excess of reactive dye over enzyme subunit concentration MTS was quantitatively inactivated within 20 min in the ATP/pyrophosphate exchange assay, whereas WTS and YTS show an 80% loss of activity over the same time period. The inactivation is affected by the addition of substrates, which either protect (WTS and YTS) or promote (YTS with tyrosine) the dye-mediated enzyme inactivation. Green HE-4BD-OH was shown to be a competitive inhibitor of MTS with respect to MgATP, methionine and tRNA substrates.

scholarly journals A translation-independent function of PheRS activates growth and proliferation in Drosophila

2021 ◽  
Vol 14 (3) ◽  
pp. dmm048132 ◽  
Author(s):  
Manh Tin Ho ◽  
Jiongming Lu ◽  
Dominique Brunßen ◽  
Beat Suter
Keyword(s):  
Trna Synthetase ◽  
Wing Disc ◽  
Follicle Cells ◽  
Independent Function ◽  
Proliferating Cells ◽  
Trna Synthetases ◽  
Elevated Expression ◽  
Aminoacyl Transfer ◽  
Cell Growth And Proliferation ◽  
Normal Twin

ABSTRACTAminoacyl transfer RNA (tRNA) synthetases (aaRSs) not only load the appropriate amino acid onto their cognate tRNAs, but many of them also perform additional functions that are not necessarily related to their canonical activities. Phenylalanyl tRNA synthetase (PheRS/FARS) levels are elevated in multiple cancers compared to their normal cell counterparts. Our results show that downregulation of PheRS, or only its α-PheRS subunit, reduces organ size, whereas elevated expression of the α-PheRS subunit stimulates cell growth and proliferation. In the wing disc system, this can lead to a 67% increase in cells that stain for a mitotic marker. Clonal analysis of twin spots in the follicle cells of the ovary revealed that elevated expression of the α-PheRS subunit causes cells to grow and proliferate ∼25% faster than their normal twin cells. This faster growth and proliferation did not affect the size distribution of the proliferating cells. Importantly, this stimulation proliferation turned out to be independent of the β-PheRS subunit and the aminoacylation activity, and it did not visibly stimulate translation.This article has an associated First Person interview with the joint first authors of the paper.

scholarly journals Properties and substrate specificity of the leucyl-, the threonyl- and the valyl-transfer-ribonucleic acid synthetases from Aesculus species

1970 ◽  
Vol 119 (4) ◽  
pp. 691-697 ◽  
Author(s):  
J. W. Anderson ◽  
L. Fowden
Keyword(s):  
Substrate Specificity ◽  
Ribonucleic Acid ◽  
Trna Synthetase ◽  
The Other ◽  
Aesculus Hippocastanum ◽  
Alternative Substrates ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Exchange Technique

1. Leucyl- and threonyl-tRNA synthetases were partially purified up to 100-fold and 30-fold respectively from cotyledons of Aesculus hippocastanum and were largely separated from the other aminoacyl-tRNA synthetases. Valyl-tRNA synthetase was purified 25-fold from cotyledons of Aesculus californica. 2. Some properties are reported for the three enzymes when assayed by the [32P]pyrophosphate-ATP exchange technique. 3. β-(Methylenecyclopropyl)alanine, isoleucine, azaleucine, norleucine and γ-hydroxynorvaline acted as alternative substrates for the leucyl-tRNA synthetase; the enzyme's affinity for β-(methylenecyclopropyl)-alanine and for isoleucine was about 80-fold less than that exhibited for leucine. 4. α-Cyclopropylglycine and α-cyclobutylglycine acted as alternative substrates for the valyl-tRNA synthetase.

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