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Information on EC 6.1.1.15 - proline-tRNA ligase and Organism(s) Methanocaldococcus jannaschii and UniProt Accession Q58635
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6.1.1.15 proline-tRNA ligaseSpecify your search results
Word Map
- 6.1.1.15
- synthetases
- aminoacylation
- halofuginone
-
ala-trnapro
-
anticodons
-
mischarged
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aarss
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misactivated
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multisynthetase
-
misacylated
-
trans-editing
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cys-trnapro
- trnacys
- thrrs
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mistranslation
- thermautotrophicus
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multi-trna
- glnrs
- leucyl-trna
- lysyl-trna
- leurs
- asprs
- febrifugine
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post-transfer
-
transcript-selective
- drug development
- pharmacology
The taxonomic range for the selected organisms is: Methanocaldococcus jannaschii
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
prors, prolyl-trna synthetase, glutamyl-prolyl-trna synthetase, glutamyl-prolyl trna synthetase, gluprors, prolyl trna synthetase, prorstt, procysrs, bifunctional aminoacyl-trna synthetase, class ii prolyl-trna synthetase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Global RNA synthesis factor
Pro-tRNA synthetase
Proline translase
Proline--tRNA ligase
Prolyl RNA synthetase
Prolyl-transfer ribonucleate synthetase
Prolyl-transfer ribonucleic acid synthetase
Prolyl-transfer RNA synthetase
Prolyl-tRNA synthetase
ProRS
Global RNA synthesis factor
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-
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Pro-tRNA synthetase
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Proline translase
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Proline--tRNA ligase
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-
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Prolyl RNA synthetase
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-
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Prolyl-transfer ribonucleate synthetase
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Prolyl-transfer ribonucleate synthetase
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Prolyl-transfer ribonucleic acid synthetase
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Prolyl-transfer ribonucleic acid synthetase
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Prolyl-transfer RNA synthetase
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Prolyl-tRNA synthetase
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Prolyl-tRNA synthetase
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ProRS
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + L-proline + tRNAPro = AMP + diphosphate + L-prolyl-tRNAPro
enzyme with dual substrate specificity which utilizes L-proline and L-cysteine as substrates, discriminating mechanism
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ATP + L-proline + tRNAPro = AMP + diphosphate + L-prolyl-tRNAPro
one tRNA binds per dimeric enzyme, only 50% of active sites are active
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
esterification
Aminoacylation
esterification
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Aminoacylation
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SYSTEMATIC NAME
IUBMB Comments
L-proline:tRNAPro ligase (AMP-forming)
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CAS REGISTRY NUMBER
COMMENTARY
9055-68-9
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + L-alanine + tRNAPro
AMP + diphosphate + L-alanyl-tRNAPro
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misacetylation of tRNAPro, no editing
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?
ATP + L-cysteine + tRNA
AMP + diphosphate + L-cysteinyl-tRNA
in later work it is shown that cysteine is attached to tRNA(Pro) and thus constitutes a misaminoacylation event and not a dual specificity
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-
?
ATP + L-cysteine + tRNA
AMP + diphosphate + L-cysteinyl-tRNA
in later work it is shown that cysteine is attached to tRNA(Pro) and thus constitutes a misaminoacylation event and not a dual specificity. Addition of tRNA has a small threefold stimulatory effect on cysteine activation. The lack of a major role of tRNA in activation of cysteine suggests that the dual specificity enzyme must distinguish cysteine from proline directly, without the assistance of each cognate tRNA, to achieve the necessary specificity required for protein synthesis
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-
?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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-
-
?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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-
-
-
?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
activation of proline does not require tRNA. The dual specificity enzyme must distinguish cysteine from proline directly, without the assistance of each cognate tRNA, to achieve the necessary specificity required for protein synthesis
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-
?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
mutations are introduced into the acceptor stem and the anticodon. Variants containing a single substitution of C72 or A73, or of one of the anticodon nucleotides, are created by site-directed mutagenesis. Substitution of A73 in Methanococcus jannaschii tRNAPro by any of the other three nucleotides has a small effect (about 10fold) on kcat/Km. The G73 substitution has the most severe effect (12.2fold), whereas the C73 or U73 substitution has a smaller effect (2-fold and 6-fold, respectively). A73 is much less significant for Methanococcus jannaschii tRNA recognition than it is for the Escherichia coli tRNA. The weak contribution of A73 to recognition by Methanococcus jannaschii ProRS, however, is comparable with that of C73 in the human system. Position 72 of Methanococcus jannaschii tRNAPro also has a minor role in aminoacylation. Substitution with any other nucleotide has a less than 10fold effect on kcat/Km. The U substitution results in the largest decrease (5.3fold), followed by the G substitution (2.2fold). The A substitution is well tolerated with only a 1.4fold decrease in kcat/Km. Substitution of U34 with A or G has no effect on aminoacylation, whereas substitution with C results in only a 1.3fold decrease. The anticodon nucleotide G35 of tRNAPro is a minor determinant for aminoacylation. Substitution of G35 with A has the largest effect (9fold), followed by the C substitution (8fold), and the U substitution (1.5fold). The most important specificity determinant in Methanococcus jannaschii tRNAPro is G36. Substitution with C alone reduces the kcat/Km of aminoacylation by 250fold, and substitution with A results in a nearly 40fold decrease. The exception is the U substitution, which maintains a kcat/Km that is similar to that of the wild-type tRNA. A representative aminoacylation assay of the C36, A36, and U36 mutants relative to that of the wild-type is shown
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-
?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
the unmodified transcript of Methanococcus jannaschii tRNAPro is mis-acylated with cysteine. The origin of mischarging is not at the anticodon or acceptor stem. Replacement of the D loop in the tRNA core with that of tRNACys suppresses mischarging with cysteine without compromising the activity of aminoacylation with proline. Prevention of mis-placement by alteration of the core structure or by nucleotide modifications in the tRNA illustrates a novel strategy of the dual-specificity synthetase
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-
?
ATP + L-cysteine + tRNAPro
AMP + diphosphate + L-cysteinyl-tRNAPro
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-
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?
ATP + L-cysteine + tRNAPro
AMP + diphosphate + L-cysteinyl-tRNAPro
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two-step reaction
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r
ATP + L-cysteine + tRNAPro
AMP + diphosphate + L-cysteinyl-tRNAPro
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misacetylation of tRNAPro, no editing
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?
ATP + L-cysteine + tRNAPro
AMP + diphosphate + L-cysteinyl-tRNAPro
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misacetylation, no editing of the misactivated Cys-tRNAPro
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?
ATP + L-cysteine + tRNAPro
AMP + diphosphate + L-cysteinyl-tRNAPro
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two-step reaction, both steps are dependent on tRNACys
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r
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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-
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?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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-
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?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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-
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?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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r
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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two-step reaction
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r
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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the enzyme mischarges tRNAPro with alanine at a rate that is 6800fold reduced relative to charging with proline. The enzyme is able to hydrolyze misactivated alanine via both pretransfer and post-transfer editing pathways. Mischarging of a tRNAPro transcript with cysteine is also detected. The enzyme stimulates ATP hydrolysis activity with the noncognate amino acid alanine but not in the presence of proline
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?
additional information
?
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enzyme does not perform deacetylation reaction of mischarged Cys-tRNAPro and Ala-tRNAPro, the enzyme also performs the ATP-diphosphate exchange reaction, no charging of tRNACys with L-cysteine
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?
additional information
?
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the enzyme also performs the ATP-diphosphate exchange reaction
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?
additional information
?
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the enzyme also performs the ATP-diphosphate exchange reaction, the enzyme possesses a pre- and post-editing mechanism for alanine
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?
additional information
?
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aminoacylation specificity, the archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNAPro with proline or cysteine, but does not aminoacylate archaeal tRNACys with cysteine, overview
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?
additional information
?
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determination of Pro-AMP and Ala-AMP hydrolysis activities, overview
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?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + L-cysteine + tRNAPro
AMP + diphosphate + L-cysteinyl-tRNAPro
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two-step reaction
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r
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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-
-
?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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-
-
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?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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-
-
?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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-
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?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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-
-
?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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-
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?
ATP + L-proline + tRNAPro
AMP + diphosphate + L-prolyl-tRNAPro
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r
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
L-cysteine
competitive inhibition of prolylation. A 40fold excess over L-proline concentration reduces the prolylation activity by 80%, no inhibition of mutant P100A
L-proline
competitive inhibition of cysteinylation. A 40fold excess over L-cysteine concentration reduces the cysteinylation activity by over 80%, no inhibition of mutant E103A
D-cysteine-DL-homocysteine
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inhibition of Cys-tRNAPro formation
additional information
-
no inhibition of Cys-tRNAPro formation by L-sulfinic acid, L-cysteic acid, S-methyl L-cysteine, and homocysteine thiolactone
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.022
L-cysteine
aminoacylation reaction, recombinant wild-type enzyme, pH 7.0, 70°C
0.027
L-proline
aminoacylation reaction, recombinant wild-type enzyme, pH 7.0, 70°C
0.049
ATP
aminoacylation reaction with L-proline, recombinant wild-type enzyme, pH 7.0, 70°C
0.06
ATP
aminoacylation reaction with L-cysteine, recombinant wild-type enzyme, pH 7.0, 70°C
0.09
L-cysteine
-
ATP-diphosphate exchange reaction, pH 7.5, 35°C
0.012
L-proline
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60°C, pH not specified in the publication, ATP-diphosphate exchange assay
0.285
L-proline
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ATP-diphosphate exchange reaction, pH 7.5, 35°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.02
L-cysteine
aminoacylation reaction, recombinant wild-type enzyme, pH 7.0, 70°C
0.09
L-proline
aminoacylation reaction, recombinant wild-type enzyme, pH 7.0, 70°C
210
L-proline
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60°C, pH not specified in the publication, ATP-diphosphate exchange assay
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
12000
L-proline
-
60°C, pH not specified in the publication, ATP-diphosphate exchange assay
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.2
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
vapour-diffusion method, protein solution is mixed with an equal volume of well solution containing 0.2 M ammonium sulfate, 20-30% PEG 4000, 5 mM 2-mercaptoethanol, 0.1 M sodium acetate, pH 4.5, 12-25°C, a few days, X-ray diffraction structure determination at 3.2 A resolution, and analysis
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
E103A
unaltered thermostability, no remaining prolylation activity, 5% remaining cysteinylation activity compared to the wild-type enzyme
P100A
unaltered thermostability, loss of 90% cysteinylation activity, unaltered prolylation activity compared to the wild-type enzyme
additional information
additional information
-
the C-terminally truncated enzyme is 3fold less active with L-cysteine and 10fold less active with L-alanine compared too the wild-type enzyme
additional information
-
C-terminal deletion mutant lacking last 50 amino acids, little effect on kinetic parameters
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged enzyme from expression in Escherichia coli
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recombinant His-tagged enzyme from overexpression in Escherichia coli
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recombinant His-tagged wild-type and C-terminally truncated enzymes from overexpressing Escherichia coli
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of the His-tagged enzyme in Escherichia coli BL21(DE3)
recombinant His-tagged wild-type and mutants from overexpressing Escherichia coli BL21
overexpression of wild-type and C-terminally truncated enzymes as His-tagged proteins in Escherichia coli
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Stathopoulos, C.; Jacquin-Becker, C.; Becker, H.D.; Li, T.; Ambrogelly, A.; Longman, R.; Soll, D.
Methanococcus jannaschii prolyl-cysteinyl-tRNA synthetase possesses overlapping amino acid binding sites
Biochemistry
40
46-52
2001
Methanocaldococcus jannaschii (Q58635), Methanocaldococcus jannaschii, Methanocaldococcus jannaschii DSM 2661 (Q58635)
Jacquin-Becker, C.; Ahel, I.; Ambrogelly, A.; Ruan, B.; Soll, D.; Stathopoulos, C.
Cysteinyl-tRNA formation and prolyl-tRNA synthetase
FEBS Lett.
514
34-36
2002
Thermus thermophilus, Giardia intestinalis, Methanocaldococcus jannaschii, Methanococcus maripaludis
Burke, B.; Lipman, R.S.A.; Shiba, K.; Musier-Forsyth, K.; Hou, Y.M.
Divergent adaptation of tRNA recognition by Methanococcus jannaschii prolyl-tRNA synthetase
J. Biol. Chem.
276
20286-20291
2001
Methanocaldococcus jannaschii (Q58635), Methanocaldococcus jannaschii, Methanocaldococcus jannaschii DSM 2661 (Q58635)
Beuning, P.J.; Musier-Forsyth, K.
Species-specific differences in amino acid editing by class II prolyl-tRNA synthetase
J. Biol. Chem.
276
30779-30785
2001
Escherichia coli, Homo sapiens, Methanocaldococcus jannaschii
Ahel, I.; Stathopoulos, C.; Ambrogelly, A.; Sauerwald, A.; Toogood, H.; Hartsch, T.; Soll, D.
Cysteine activation is an inherent in vitro property of prolyl-tRNA synthetases
J. Biol. Chem.
277
34743-34748
2002
Aquifex aeolicus, Borreliella burgdorferi, Saccharomyces cerevisiae, Acetoanaerobium sticklandii, Deinococcus radiodurans, Escherichia coli, Thermus thermophilus, Magnetospirillum magnetotacticum, Methanothermobacter thermautotrophicus, Methanocaldococcus jannaschii, Rhodopseudomonas palustris, Novosphingobium aromaticivorans, Cytophaga hutchinsonii
Ambrogelly, A.; Ahel, I.; Polycarpo, C.; Bunjun-Srihari, S.; Krett, B.; Jacquin-Becker, C.; Ruan, B.; Kohrer, C.; Stathopoulos, C.; RajBhandary, U.L.; Soll, D.
Methanocaldococcus jannaschii prolyl-tRNA synthetase charges tRNA(Pro) with cysteine
J. Biol. Chem.
277
34749-34754
2002
Methanocaldococcus jannaschii
Kamtekar, S.; Kennedy, W.D.; Wang, J.; Stathopoulos, C.; Soll, D.; Steitz, T.A.
The structural basis of cysteine aminoacylation of tRNAPro by prolyl-tRNA synthetases
Proc. Natl. Acad. Sci. USA
100
1673-1678
2003
Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus
Ambrogelly, A.; Kamtekar, S.; Stathopoulos, C.; Kennedy, D.; Soll, D.
Asymmetric behavior of archaeal prolyl-tRNA synthetase
FEBS Lett.
579
6017-6022
2005
Methanothermobacter thermautotrophicus, Methanocaldococcus jannaschii
Hati, S.; Ziervogel, B.; Sternjohn, J.; Wong, F.C.; Nagan, M.C.; Rosen, A.E.; Siliciano, P.G.; Chihade, J.W.; Musier-Forsyth, K.
Pre-transfer editing by class II prolyl-tRNA synthetase: role of aminoacylation active site in 'selective release' of noncognate amino acids
J. Biol. Chem.
281
27862-27872
2006
Saccharomyces cerevisiae, Escherichia coli, Methanothermobacter thermautotrophicus, Methanocaldococcus jannaschii, Methanococcus maripaludis
Splan, K.E.; Ignatov, M.E.; Musier-Forsyth, K.
Transfer RNA modulates the editing mechanism used by class II prolyl-tRNA synthetase
J. Biol. Chem.
283
7128-7134
2008
Escherichia coli, Homo sapiens, Methanocaldococcus jannaschii
Koehrer, C.; Rajbhandary, U.L.
The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases
Methods
44
129-138
2008
Methanothermobacter thermautotrophicus, Methanocaldococcus jannaschii, Methanopyrus kandleri
Lipman, R.S.; Wang, J.; Sowers, K.R.; Hou, Y.M.
Prevention of mis-aminoacylation of a dual-specificity aminoacyl-tRNA synthetase
J. Mol. Biol.
315
943-999
2002
Methanocaldococcus jannaschii (Q58635), Methanocaldococcus jannaschii, Methanocaldococcus jannaschii DSM 2661 (Q58635)
Lipman, R.S.; Beuning, P.J.; Musier-Forsyth, K.; Hou, Y.M.
Amino acid activation of a dual-specificity tRNA synthetase is independent of tRNA
J. Mol. Biol.
316
421-427
2002
Methanocaldococcus jannaschii (Q58635), Methanocaldococcus jannaschii, Methanocaldococcus jannaschii DSM 2661 (Q58635)
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