Literature summary for 5.4.99.12 extracted from

Hur, S.; Stroud, R.M.
How U38, 39, and 40 of many tRNAs become the targets for pseudouridylation by TruA (2007), Mol. Cell, 26, 189-203.

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Cloned(Commentary)

Cloned (Comment)	Organism
-	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
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	Escherichia coli
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	Escherichia coli

Crystallization (Comment)

Organism

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

Escherichia coli

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

Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
R58A	inactive	Escherichia coli
R58A	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. The wild-type and mutant enzymes have similar thermal stabilities based on identical tryptophan fluorescence curves over the range of melting temperatures, indicating that the R58A mutation does not drastically perturb the enzyme structure	Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
tRNA uridine38-40	Escherichia coli	TruA specifically modifies uridines at positions 38, 39, and/or 40 of tRNAs with highly divergent sequences and structures	tRNA pseudouridine38-40	-	?

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	-	-	-
Escherichia coli	P07649	-	-

Reaction

Reaction	Comment	Organism	Reaction ID
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	Escherichia coli	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
tRNA uridine38	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	Escherichia coli	tRNA pseudouridine38	-	?
tRNA uridine38-40	TruA specifically modifies uridines at positions 38, 39, and/or 40 of tRNAs with highly divergent sequences and structures	Escherichia coli	tRNA pseudouridine38-40	-	?
tRNA uridine38-40	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	Escherichia coli	tRNA pseudouridine38-40	-	?
tRNA uridine39	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	Escherichia coli	tRNA pseudouridine39	-	?
tRNA uridine40	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	Escherichia coli	tRNA pseudouridine40	-	?
tRNALeu3	-	Escherichia coli	?	-	?
tRNALeu3 carrying uridine at position 38	-	Escherichia coli	tRNALeu3 carrying pseudouridine at position 38	-	?
tRNALeu3 carrying uridine at position 39	-	Escherichia coli	tRNALeu3 carrying pseudouridine at position 39	-	?
tRNALeu3 carrying uridine at position 40	-	Escherichia coli	tRNALeu3 carrying pseudouridine at position 40	-	?

Synonyms

Synonyms	Comment	Organism
pseudouridine synthase	-	Escherichia coli
TruA	-	Escherichia coli

General Information

General Information	Comment	Organism
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	Escherichia coli
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	Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.16	-	tRNALeu3	pH 8.0, 22°C	Escherichia coli
0.2	-	tRNALeu3 carrying uridine at position 39	pH 8.0, 22°C	Escherichia coli
0.86	-	tRNALeu3 carrying uridine at position 40	pH 8.0, 22°C	Escherichia coli
160	-	tRNA uridine38	pH 8.0, wild-type enzyme	Escherichia coli
200	-	tRNA uridine39	pH 8.0, wild-type enzyme	Escherichia coli
860	-	tRNA uridine40	pH 8.0, wild-type enzyme	Escherichia coli