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Information on EC 5.4.99.12 - tRNA pseudouridine38-40 synthase
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5.4.99.12 tRNA pseudouridine38-40 synthaseIUBMB Comments
The uridylate residues at positions 38, 39 and 40 of nearly all tRNAs are isomerized to pseudouridine. TruA specifically modifies uridines at positions 38, 39, and/or 40 in the anticodon stem loop of tRNAs with highly divergent sequences and structures .
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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Reaction Schemes
tRNA uridine38-40
=
tRNA pseudouridine38-40
Synonyms
trna pseudouridine synthase i, mpus3p, pus3p, deg1p, pseudouridine synthase 3, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
hisT
pseudouridine synthase I
tRNA pseudouridine synthase I
TruA
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
tRNA uridine38-40 = tRNA pseudouridine38-40
chemical evidence against a covalent cysteine intermediate in the rearrangement mechanism of uridine to pseudouridine
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tRNA uridine38-40 = tRNA pseudouridine38-40
structural, computational, and functional studies provide the basis for a substrate recognition model for the regional selectivity. By binding to the conserved parts of tRNAs (elbow and D stem backbone), TruA recognizes multiple tRNAs independent of sequence variations. Anchored at these two regions, TruA positions the anticodon stem loop near the active site without constraining its flexibility, thereby increasing the effective concentration of each target position, 38, 39, and 40, in the vicinity of the active site. The thermal motions of the anticodon stem loop allow the nucleotides at each of the three sites to be dynamically accessible for modification. TruA utilizes the intrinsic flexibility of the anticodon stem loop for site promiscuity and also to select against intrinsically stable tRNAs
tRNA uridine38-40 = tRNA pseudouridine38-40
the mechanism of pseudouridine synthase I is deduced from its interaction with 5-fluorouracil-tRNA. The covalent complex formed between pseudouridine synthase I and 5-fluorouracil-tRNA involves Michael adduct formation between Asp60 of pseudouridine synthase I and the 6-carbon of 5-fluorouracil39 of tRNA to form a covalent pseudouridine synthase I-5-fluorouracil-tRNA complex
tRNA uridine38-40 = tRNA pseudouridine38-40
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SYSTEMATIC NAME
IUBMB Comments
tRNA-uridine38-40 uracil mutase
The uridylate residues at positions 38, 39 and 40 of nearly all tRNAs are isomerized to pseudouridine. TruA specifically modifies uridines at positions 38, 39, and/or 40 in the anticodon stem loop of tRNAs with highly divergent sequences and structures [1].
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CAS REGISTRY NUMBER
COMMENTARY
430429-15-5
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61506-89-6
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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
Escherichia coli tRNAPhe uridine39
Escherichia coli tRNAPhe pseudouridine39
-
-
-
-
?
human tRNALeu uridine 38 uridine39
human tRNALeu pseudouridine 38 pseudouridine39
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psuedouridine formation in position 39 is clearly preferred over position 38
-
?
Salmonella typhimurium HisT- tRNALeu
?
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tRNAPhe2 from mutant HisT- strain defective for tRNA pseudouridine synthase I that forms pseudouridine in the 3'-side of the anticodon region of approximately half of the cellular tRNAs
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-
?
Salmonella typhimurium HisT- tRNAPhe2 uridine39
Salmonella typhimurium HisT- tRNAPhe2 pseudouridine39
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tRNAPhe2 from mutant HisT- strain defective for tRNA pseudouridine synthase I that forms pseudouridine in the 3 side of the anticodon region of approximately half of the cellular tRNAs
specific modification of uridine39
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?
Salmonelly typhimurium HisT- tRNATyr
?
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tRNAPhe2 from mutant HisT- strain defective for tRNA pseudouridine synthase I that forms pseudouridine in the 3'-side of the anticodon region of approximately half of the cellular tRNAs
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-
?
tRNA uridine40
tRNA pseudouridine40
modified tRNALeu3 with uridine at position 40 instead of position 38. Wild-typeTruA pseudouridylates uridines at all three positions (38, 39 and 40) with efficiencies (kcat/KM) differing by less than 10fold, while R58A is inactive toward all three uridines
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-
?
tRNAHis guanidine36 uridine38 cytidine39
RNAHis guanidine36 pseudouridine38 cytidine39
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-
-
-
?
tRNAHis uridine38 uridine39
tRNAHis pseudouridine38 pseudouridine39
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-
-
-
?
tRNALeu3 carrying uridine at position 38
tRNALeu3 carrying pseudouridine at position 38
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-
-
?
tRNALeu3 carrying uridine at position 39
tRNALeu3 carrying pseudouridine at position 39
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-
-
?
tRNALeu3 carrying uridine at position 40
tRNALeu3 carrying pseudouridine at position 40
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-
-
?
tRNAVal2a cytidine36 uridine38
tRNAVal2a cytidine36 pseudouridine38
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poor substrate, tRNAVal2a is not a substrate
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-
?
additional information
?
-
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presence of a G36 residue modulates modification at position 38. In addition to local sequence effects, steady-state kinetic analyses suggest the existence of other recognition elements distinct from the immediate vicinity of modification
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-
?
tRNA uridine38
tRNA pseudouridine38
tRNALeu3 contains uridine at position 38. Wild-typeTruA pseudouridylates uridines at all three positions (38, 39 and 40) with efficiencies (kcat/KM) differing by less than 10fold, while R58A is inactive toward all three uridines. When flexibility of the anticodon stem loop is increased by mutating the two G:C base pairs in the stem of the anticodon stem loop of tRNALeu3 into A:U pairs, the kcat/KM increased 2fold. When flexibility is decreased by base-pairing the target U38 of tRNALeu3 with A32 instead of with U32, the kcat/KM decreases 10fold
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-
?
tRNA uridine38
tRNA pseudouridine38
the enzyme modifies the anticodon arm of transfer RNA at positions 38 and 39 by catalyzing the conversion of uridine to pseudouridine
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-
?
tRNA uridine38-40
tRNA pseudouridine38-40
TruA specifically modifies uridines at positions 38, 39, and/or 40 of tRNAs with highly divergent sequences and structures
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-
?
tRNA uridine38-40
tRNA pseudouridine38-40
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purified tRNA pseudouridine synthase I modifies all of the hisT isoacceptors of tRNAHis, tRNATyr, and tRNALeu to products which are chromatographically indistinguishable from the respective wild-type species. These three groups of isoacceptors contain all the known topological sites for pseudouridine modification of residues 38,39, and 40
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-
?
tRNA uridine38-40
tRNA pseudouridine38-40
TruA specifically modifies uridines at positions 38, 39, and/or 40 of tRNAs with highly divergent sequences and structures. The molecular basis for the site and substrate promiscuity is studied by determining the crystal structures of Eschrichia coli TruA in complex with two different leucyl tRNAs in conjunction with functional assays and computer simulation
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-
?
tRNA uridine39
tRNA pseudouridine39
modified tRNALeu3 with uridine at position 39 instead of position 38. Wild-typeTruA pseudouridylates uridines at all three positions (38, 39 and 40) with efficiencies (kcat/KM) differing by less than 10fold, while R58A is inactive toward all three uridines
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-
?
tRNA uridine39
tRNA pseudouridine39
tRNAPhe from yeast, which contains a single target for tRNA pseudouridine synthase I at U39
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?
tRNA uridine39
tRNA pseudouridine39
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tRNAPhe with uridine at position 39
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-
?
tRNA uridine39
tRNA pseudouridine39
with tRNA substrates from both yeast and humans, uridines at position 39 are modified to pseudouridine. In a tRNA substrate with a uridine at position 38 (human tRNALeu), there is very slight formation of pseudouridine at that position after incubation with mPus3p
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-
?
tRNA uridine39
tRNA pseudouridine39
the enzyme modifies the anticodon arm of transfer RNA at positions 38 and 39 by catalyzing the conversion of uridine to pseudouridine
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-
?
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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
tRNA uridine38
tRNA pseudouridine38
the enzyme modifies the anticodon arm of transfer RNA at positions 38 and 39 by catalyzing the conversion of uridine to pseudouridine
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-
?
tRNA uridine38-40
tRNA pseudouridine38-40
TruA specifically modifies uridines at positions 38, 39, and/or 40 of tRNAs with highly divergent sequences and structures
-
-
?
tRNA uridine39
tRNA pseudouridine39
with tRNA substrates from both yeast and humans, uridines at position 39 are modified to pseudouridine. In a tRNA substrate with a uridine at position 38 (human tRNALeu), there is very slight formation of pseudouridine at that position after incubation with mPus3p
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-
?
tRNA uridine39
tRNA pseudouridine39
the enzyme modifies the anticodon arm of transfer RNA at positions 38 and 39 by catalyzing the conversion of uridine to pseudouridine
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-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
fluorouracil-substituted tRNA
causes a time-dependent inactivation of pseudouridine synthase I and forms a covalent complex with the enzyme that involves the fluorouracil-substituted UMP at position 39. Upon incubation of 100 nM pseudouridine synthase with 0.001 mM fluorouracil-substituted tRNA at 15°C prior to addition of substrate, there is a time-dependent inactivation of the enzyme with a half-life of 35 min
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tRNA
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activity is inhibited by single tRNAs or bulk tRNA from all sources tested, and by ribosomal RNA
5-fluorouracil tRNAPhe
time-dependent inactivation of pseudouridine synthase I and formation of a covalent complex with the enzyme that involves the 5-fluorouracil monophosphate at position 39
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5-fluorouracil tRNAPhe
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enzyme forms a covalent adduct with 5-fluorouracil-tRNA to form a putative analog of a steady-state intermediate in the normal reaction pathway. The putative Asp nucleophile is attached to the 6-position of the target 5-fluorouracil-tRNA to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage, and the intermediate contains an intact N-glycosidic bond linking the modified base to the polynucleotide chain
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.00011
Escherichia coli tRNAPhe uridine39
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mutant C55A/C154A/C169A, pH 8.0, 30°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
physiological function
malfunction
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hisT mutants of Salmonella typhimurium lack the enzyme that modifies uridine to pseudouridine in the anticodon regions of many tRNAs. The regulation of a large number of amino acid biosynthetic pathways is altered by the hisT mutation
malfunction
tRNA pseudouridine synthase is able to complement the type III gene expression defect of the fimV mutant. Thus fimV and truA form an operon and fimV mutation has a polar effect on truA. A truA mutant is defective in type III gene expression while its twitching motility is unaffected, and a truA clone is able to complement the type III secretion defect
malfunction
deletion of the PUS3 gene has an effect on the efficiency of the translation process. Reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs
malfunction
disruption of the DEG1 gene is not lethal but reduces considerably the yeast growth rate, especially at an elevated temperature, 37 °C
physiological function
the truA gene of Pseudomonas aeruginosa is required for the expression of type III secretory genes
physiological function
TruA utilizes the intrinsic flexibility of the ASL for site promiscuity and also to select against intrinsically stable tRNAs to avoid their overstabilization through pseudouridylation, thereby maintaining the balance between the flexibility and stability required for its biological function
physiological function
deletion of the PUS3 gene, encoding the enzyme that introduces pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process. In the mutant, there is a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs. This effect is solely due to the absence of pseudouridine 38 or 39 in tRNA. The presence of pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast
physiological function
TruA utilizes the intrinsic flexibility of the anticodon stem loop for site promiscuity and also to select against intrinsically stable tRNAs to avoid their overstabilization through pseudouridylation, thereby maintaining the balance between the flexibility and stability required for its biological function
physiological function
Pus3p is unique in its ability to modulate frameshifting and readthrough events during translation. This aspect of its activity may be responsible for HOT1 recombination phenotypes observed in deg1 mutants
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
31000
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homodimer
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structures of TruA in complex with two different leucyl-tRNAs to 3.5-4.0 A resolution, in conjunction with functional assays and computer simulation. The structures capture three stages of the TruA-tRNA reaction, TruA utilizes the intrinsic flexibility of the anticodon stem loop for site promiscuity and also to select against intrinsically stable tRNAs to avoid their overstabilization through pseudouridylation, thereby maintaining the balance between the flexibility and stability required for its biological function
hanging-drop vapor diffusion method at room temperature. It is attempted to obtain structures of Escherichia coli TruA complexed with three Escherichia coli tRNAs representing all of the target sites: tRNALeu1 with uridine at 39, tRNALeu2 with uridine at 38 and 40, and tRNALeu3 with uridine at 38. These tRNAs are type II tRNAs with a 15 nucleotide variable loop. Three crystal forms are obtained from similar buffer conditions, containing the complex of the wild-type TruA and full-length tRNALeu1 in crystal I, and the complex of wild-type TruA and tRNALeu3 in crystal forms II and III. No crystals are obtained with tRNALeu2
native protein, to 1.5 A resolution, and several derivatives. Structure reveals a dimeric protein that contains two positively charged, RNA-binding clefts along the surface of the protein. Each cleft contains a highly conserved aspartic acid located at its center. The structure suggests that a dimeric enzyme is required for binding transfer RNA and subsequent pseudouridine formation
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hanging-drop vapor diffusion method at 293 K. The crystals have a stick-like shape, and belong to the space group P4(1)2(1)2, with unit cell dimensions of a = b = 91.5 A and c = 164.0 A. Crystal structure is determined at 2.25 A resolution
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to 2.25 A resolution. Structure reveals the remarkably flexible structural features in the tRNA-binding cleft, which may be responsible for the primary tRNA interaction. The charged residues occupying the intermediate positions in the cleft may lead the tRNA to the active site for catalysis. The tRNA probably makes the melting base pairs move into the cleft, and a conformational change of the substrate tRNA may be necessary to facilitate access to the active site aspartate residue, deep within the cleft
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C154A
C154S
C169A
C169S
C55A/C154A/C169A
C55S
D60A
D60E
D60K
D60N
D60S
R58A
D151A
additional information
C154S
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complete loss of activity, mutant is capable of binding to substrate tRNAPhe
C169S
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complete loss of activity, mutant is unable to bind substrate tRNAPhe
C55A/C154A/C169A
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only a small change in both Km and Vmax parameters as compared with wild-type enzyme. These mutations cause a 1.2fold increase in Km and a 1.6fold decrease in Vmax. The overall Vmax/Km ratio is lowered by a factor of 2 for the triple mutant
D60A
mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
D60A
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA
D60E
mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
D60E
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA
D60K
mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
D60K
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA
D60N
mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
D60N
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA
D60S
mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
D60S
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA