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 .
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
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
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
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].
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
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
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
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
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
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
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
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
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
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
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
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
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
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
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
all Asp60 mutants bind tRNA but are catalytically inactive and fail to form covalent complexes with fluorouracil-substituted tRNA. It is concluded that the conserved Asp60 is essential for pseudouridine synthase activity
all Asp60 mutants bind tRNA but are catalytically inactive and fail to form covalent complexes with fluorouracil-substituted tRNA. It is concluded that the conserved Asp60 is essential for pseudouridine synthase activity
isolation of virtually homogeneous tRNA pseudouridine synthase I from strains of Escherichia coli transformed with plasmid in which the production of tRNA pseudouridine synthase I is amplified 20fold