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1.
J Biochem ; 130(6): 727-30, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11726270

ABSTRACT

At the initiation of chromosomal DNA replication, DNA primases synthesize short RNA primers, which are subsequently elongated by DNA polymerases. To understand the structural basis for the primer synthesis by archaeal/eukaryotic-type primases, the gene of the DNA primase from hyperthermophilic archaeon Pyrococcus horikoshii was cloned and overexpressed in Escherichia coli as a fusion protein with a hexa-histidine tag at its amino terminus. The recombinant DNA primase was purified and crystallized by the hanging-drop vapor diffusion method at 293 K, with polyethylene glycol 8000 as the precipitant. The crystals belong to the P3(2)21 space group with unit-cell parameters a = b = 77.8, c = 129.6 A, and alpha = beta = 90 degrees, gamma = 120 degrees. Crystals of the selenomethionine derivative were obtained by means of a cross-seeding method using native crystals. The data for the native and selenomethionine-substituted crystals were collected to 1.8 and 2.2 A resolution, respectively, with synchrotron radiation at SPring-8 under flash-frozen conditions at 100 K. The four wavelength MAD data provided a phase to determine the structure of the primase at 2.2 A resolution.


Subject(s)
Crystallography, X-Ray/methods , DNA Primase/chemistry , Pyrococcus/enzymology , Amino Acid Substitution , Crystallization , DNA Primase/isolation & purification , DNA Primase/metabolism , Protein Conformation , RNA/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Selenomethionine/chemistry
2.
EMBO J ; 20(23): 6612-8, 2001 Dec 03.
Article in English | MEDLINE | ID: mdl-11726497

ABSTRACT

The human centromere protein B (CENP-B), one of the centromere components, specifically binds a 17 bp sequence (the CENP-B box), which appears in every other alpha-satellite repeat. In the present study, the crystal structure of the complex of the DNA-binding region (129 residues) of CENP-B and the CENP-B box DNA has been determined at 2.5 A resolution. The DNA-binding region forms two helix-turn-helix domains, which are bound to adjacent major grooves of the DNA. The DNA is kinked at the two recognition helix contact sites, and the DNA region between the kinks is straight. Among the major groove protein-bound DNAs, this 'kink-straight-kink' bend contrasts with ordinary 'round bends' (gradual bending between two protein contact sites). The larger kink (43 degrees ) is induced by a novel mechanism, 'phosphate bridging by an arginine-rich helix': the recognition helix with an arginine cluster is inserted perpendicularly into the major groove and bridges the groove through direct interactions with the phosphate groups. The overall bending angle is 59 degrees, which may be important for the centromere-specific chromatin structure.


Subject(s)
Autoantigens , Chromosomal Proteins, Non-Histone/chemistry , Crystallography, X-Ray , DNA-Binding Proteins , DNA/chemistry , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Centromere/metabolism , Centromere Protein B , Chromatin/chemistry , Chromatin/metabolism , Escherichia coli/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry
3.
Structure ; 9(12): 1253-63, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11738050

ABSTRACT

BACKGROUND: The AU binding homolog of enoyl-CoA hydratase (AUH) is a bifunctional protein that has two distinct activities: AUH binds to RNA and weakly catalyzes the hydration of 2-trans-enoyl-coenzyme A (enoyl-CoA). AUH has no sequence similarity with other known RNA binding proteins, but it has considerable sequence similarity with enoyl-CoA hydratase. A segment of AUH, named the R peptide, binds to RNA. However, the mechanism of the RNA binding activity of AUH remains to be elucidated. RESULTS: We determined the crystal structure of human AUH at 2.2 A resolution. AUH adopts the typical fold of the enoyl-CoA hydratase/isomerase superfamily and forms a hexamer as a dimer of trimers. Interestingly, the surface of the AUH hexamer is positively charged, in striking contrast to the negatively charged surfaces of the other members of the superfamily. Furthermore, wide clefts are uniquely formed between the two trimers of AUH and are highly positively charged with the Lys residues in alpha helix H1, which is located on the edge of the cleft and contains the majority of the R peptide. A mutational analysis showed that the lysine residues in alpha helix H1 are essential to the RNA binding activity of AUH. CONCLUSIONS: Alpha helix H1 exposes a row of Lys residues on the solvent-accessible surface. These characteristic Lys residues are named the "lysine comb." The distances between these Lys residues are similar to those between the RNA phosphate groups, suggesting that the lysine comb may continuously bind to a single-stranded RNA. The clefts between the trimers may provide spaces sufficient to accommodate the RNA bases.


Subject(s)
Enoyl-CoA Hydratase/chemistry , RNA-Binding Proteins/chemistry , Amino Acid Sequence , Binding Sites , Catalysis , Crystallography, X-Ray , DNA Mutational Analysis , DNA, Complementary/metabolism , Dimerization , Glutathione Transferase/metabolism , Humans , Lysine/chemistry , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Structure, Tertiary , RNA/metabolism , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid
4.
Proc Natl Acad Sci U S A ; 98(24): 13537-42, 2001 Nov 20.
Article in English | MEDLINE | ID: mdl-11698642

ABSTRACT

Arginyl-tRNA synthetase (ArgRS) recognizes two major identity elements of tRNA(Arg): A20, located at the outside corner of the L-shaped tRNA, and C35, the second letter of the anticodon. Only a few exceptional organisms, such as the yeast Saccharomyces cerevisiae, lack A20 in tRNA(Arg). In the present study, we solved the crystal structure of a typical A20-recognizing ArgRS from Thermus thermophilus at 2.3 A resolution. The structure of the T. thermophilus ArgRS was found to be similar to that of the previously reported S. cerevisiae ArgRS, except for short insertions and a concomitant conformational change in the N-terminal domain. The structure of the yeast ArgRS.tRNA(Arg) complex suggested that two residues in the unique N-terminal domain, Tyr(77) and Asn(79), which are phylogenetically invariant in the ArgRSs from all organisms with A20 in tRNA(Arg)s, are involved in A20 recognition. However, in a docking model constructed based on the yeast ArgRS.tRNA(Arg) and T. thermophilus ArgRS structures, Tyr(77) and Asn(79) are not close enough to make direct contact with A20, because of the conformational change in the N-terminal domain. Nevertheless, the replacement of Tyr(77) or Asn(79) by Ala severely reduced the arginylation efficiency. Therefore, some conformational change around A20 is necessary for the recognition. Surprisingly, the N79D mutant equally recognized A20 and G20, with only a slight reduction in the arginylation efficiency as compared with the wild-type enzyme. Other mutants of Asn(79) also exhibited broader specificity for the nucleotide at position 20 of tRNA(Arg). We propose a model of A20 recognition by the ArgRS that is consistent with the present results of the mutational analyses.


Subject(s)
Arginine-tRNA Ligase/chemistry , Nucleic Acid Conformation , RNA, Transfer, Arg/chemistry , Amino Acids , Arginine-tRNA Ligase/metabolism , Binding Sites , Crystallography, X-Ray , Models, Molecular , Mutagenesis , Protein Conformation , RNA, Transfer, Arg/metabolism , Saccharomyces cerevisiae/enzymology , Thermus thermophilus/enzymology
6.
Acta Crystallogr D Biol Crystallogr ; 57(Pt 11): 1659-62, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11679736

ABSTRACT

The archaeosine tRNA-guanine transglycosylase from the hyperthermophilic archaeon Pyrococcus horikoshii was crystallized and preliminary X-ray characterization was performed. Single crystals were grown by the hanging-drop vapour-diffusion method, using sodium/potassium phosphate and sodium acetate as precipitants. The space group is P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 99.28 (14), c = 363.74 (56) A. The cryocooled crystals diffracted X-rays beyond 2.2 A resolution using synchrotron radiation from station BL44XU at SPring-8 (Harima). Selenomethionine-substituted protein crystals were prepared in order to solve the structure by the MAD phasing method.


Subject(s)
Pentosyltransferases/chemistry , Pyrococcus/enzymology , Crystallization , Crystallography, X-Ray , Protein Conformation , Selenomethionine/chemistry
7.
J Biol Chem ; 276(50): 47387-93, 2001 Dec 14.
Article in English | MEDLINE | ID: mdl-11584022

ABSTRACT

An analogue of isoleucyl-adenylate (Ile-AMS) potently inhibits the isoleucyl-tRNA synthetases (IleRSs) from the three primary kingdoms, whereas the antibiotic mupirocin inhibits only the eubacterial and archaeal IleRSs, but not the eukaryotic enzymes, and therefore is clinically used against methicillin-resistant Staphylococcus aureus. We determined the crystal structures of the IleRS from the thermophilic eubacterium, Thermus thermophilus, in complexes with Ile-AMS and mupirocin at 3.0- and 2.5-A resolutions, respectively. A structural comparison of the IleRS.Ile-AMS complex with the adenylate complexes of other aminoacyl-tRNA synthetases revealed the common recognition mode of aminoacyl-adenylate by the class I aminoacyl-tRNA synthetases. The Ile-AMS and mupirocin, which have significantly different chemical structures, are recognized by many of the same amino acid residues of the IleRS, suggesting that the antibiotic inhibits the enzymatic activity by blocking the binding site of the high energy intermediate, Ile-AMP. In contrast, the two amino acid residues that concomitantly recognize Ile-AMS and mupirocin are different between the eubacterial/archaeal IleRSs and the eukaryotic IleRSs. Mutagenic analyses revealed that the replacement of the two residues significantly changed the sensitivity to mupirocin.


Subject(s)
Adenosine Monophosphate/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Isoleucine-tRNA Ligase/metabolism , Isoleucine/chemistry , Mupirocin/chemistry , Mupirocin/pharmacology , Amino Acid Sequence , Amino Acids/chemistry , Amino Acyl-tRNA Synthetases/metabolism , Binding Sites , Crystallography, X-Ray , Fatty Acids/chemistry , Inhibitory Concentration 50 , Kinetics , Models, Chemical , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphates/chemistry , Protein Conformation , Protein Structure, Secondary , Sequence Homology, Amino Acid , Staphylococcus aureus/metabolism , Thermus thermophilus
8.
Nat Struct Biol ; 8(3): 203-6, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11224561

ABSTRACT

Glutamyl-tRNA synthetases (GluRSs) are divided into two distinct types, with regard to the presence or absence of glutaminyl-tRNA synthetase (GlnRS) in the genetic translation systems. In the original 19-synthetase systems lacking GlnRS, the 'non-discriminating' GluRS glutamylates both tRNAGlu and tRNAGln. In contrast, in the evolved 20-synthetase systems with GlnRS, the 'discriminating' GluRS aminoacylates only tRNAGlu. Here we report the 2.4 A resolution crystal structure of a 'discriminating' GluRS.tRNAGlu complex from Thermus thermophilus. The GluRS recognizes the tRNAGlu anticodon bases via two alpha-helical domains, maintaining the base stacking. We show that the discrimination between the Glu and Gln anticodons (34YUC36 and 34YUG36, respectively) is achieved by a single arginine residue (Arg 358). The mutation of Arg 358 to Gln resulted in a GluRS that does not discriminate between the Glu and Gln anticodons. This change mimics the reverse course of GluRS evolution from anticodon 'non-dicsriminating' to 'discriminating'.


Subject(s)
Anticodon/chemistry , Anticodon/metabolism , Glutamate-tRNA Ligase/chemistry , Glutamate-tRNA Ligase/metabolism , Thermus thermophilus/enzymology , Thermus thermophilus/genetics , Anticodon/genetics , Binding Sites , Crystallography, X-Ray , Evolution, Molecular , Glutamate-tRNA Ligase/genetics , Glutamic Acid/metabolism , Glutamine/metabolism , Kinetics , Models, Molecular , Nucleic Acid Conformation , Point Mutation/genetics , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Transfer, Glu/chemistry , RNA, Transfer, Glu/genetics , RNA, Transfer, Glu/metabolism , Structure-Activity Relationship , Substrate Specificity
9.
Acta Crystallogr D Biol Crystallogr ; 57(Pt 2): 272-5, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11173477

ABSTRACT

The gene encoding the highly thermostable arginyl-tRNA synthetase (ArgRS) from Thermus thermophilus was cloned and overexpressed in Escherichia coli under the control of the T7 promoter. The recombinant ArgRS was purified by two chromatographic steps and was crystallized by the hanging-drop vapour-diffusion method using PEG 8000 and ethylene glycol as precipitants. The crystals belong to the hexagonal space group P6(5), with unit-cell parameters a = b = 156.04 (7), c = 87.17 (4) A. X-ray data to 2.8 A resolution were collected at room temperature from a native crystal using an in-house X-ray source. Uranium, platinum and selenomethionine derivatives were found to be useful for phasing by the multiple isomorphous replacement method with anomalous scattering. The flash-frozen crystals diffracted beyond 2.3 A resolution using synchrotron radiation from the beamline 41XU at SPring-8 (Harima).


Subject(s)
Arginine-tRNA Ligase/chemistry , Thermus thermophilus/enzymology , Amino Acid Sequence , Arginine-tRNA Ligase/genetics , Arginine-tRNA Ligase/isolation & purification , Cloning, Molecular , Escherichia coli , Ethylene Glycol , Indicators and Reagents , Molecular Sequence Data , Peptide Fragments/chemistry , Polyethylene , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Thermus thermophilus/genetics
10.
J Biol Chem ; 276(6): 3723-6, 2001 Feb 09.
Article in English | MEDLINE | ID: mdl-11106639

ABSTRACT

The presence of two short signature sequence motifs (His-Ile-Gly-His (HIGH) and Lys-Met-Ser-Lys (KMSK)) is a characteristic of the class I aminoacyl-tRNA synthetases. These motifs constitute a portion of the catalytic site in three dimensions and play an important role in catalysis. In particular, the second lysine of the KMSK motif (K2) is the crucial catalytic residue for stabilization of the transition state of the amino acid activation reaction (aminoacyl-adenylate formation). Arginyl-tRNA synthetase (ArgRS) is unique among all of the class I enyzmes, as the majority of ArgRS species lack canonical KMSK sequences. Thus, the mechanism by which this group of ArgRSs achieves the catalytic reaction is not well understood. Using three-dimensional modeling in combination with sequence analysis and site-directed mutagenesis, we found a conserved lysine in the KMSK-lacking ArgRSs upstream of the HIGH sequence motif, which is likely to be a functional counterpart of the canonical class I K2 lysine. The results suggest a plausible partition of the ArgRSs into two major groups, on the basis of the conservation of the HIGH lysine.


Subject(s)
Arginine-tRNA Ligase/metabolism , Lysine/metabolism , Amino Acid Sequence , Arginine-tRNA Ligase/chemistry , Catalysis , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino Acid
12.
Cell ; 103(5): 793-803, 2000 Nov 22.
Article in English | MEDLINE | ID: mdl-11114335

ABSTRACT

Valyl-tRNA synthetase (ValRS) strictly discriminates the cognate L-valine from the larger L-isoleucine and the isosteric L-threonine by the tRNA-dependent "double sieve" mechanism. In this study, we determined the 2.9 A crystal structure of a complex of Thermus thermophilus ValRS, tRNA(Val), and an analog of the Val-adenylate intermediate. The analog is bound in a pocket, where Pro(41) allows accommodation of the Val and Thr moieties but precludes the Ile moiety (the first sieve), on the aminoacylation domain. The editing domain, which hydrolyzes incorrectly synthesized Thr-tRNA(Val), is bound to the 3' adenosine of tRNA(Val). A contiguous pocket was found to accommodate the Thr moiety, but not the Val moiety (the second sieve). Furthermore, another Thr binding pocket for Thr-adenylate hydrolysis was suggested on the editing domain.


Subject(s)
Isoleucine/chemistry , RNA, Transfer, Val/chemistry , Threonine/chemistry , Valine-tRNA Ligase/chemistry , Valine/chemistry , Adenosine/chemistry , Binding Sites , Crystallography, X-Ray , Hydrolysis , Models, Chemical , Models, Molecular , Proline/chemistry , Protein Binding , Protein Structure, Tertiary , RNA, Transfer, Val/metabolism , Thermus thermophilus/chemistry , Valine-tRNA Ligase/metabolism
13.
J Biochem ; 127(2): 181-4, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10731682

ABSTRACT

The cell surface antigen CD38 is a multifunctional ectoenzyme that acts as an NAD(+) glycohydrolase, an ADP-ribosyl cyclase, and also a cyclic ADP-ribose hydrolase. The extracellular catalytic domain of CD38 was expressed as a fusion protein with maltose-binding protein, and was crystallized in the complex with a ganglioside, G(T1b), one of the possible physiological inhibitors of this ectoenzyme. Two different crystal forms were obtained using the hanging-drop vapor diffusion method with PEG 10,000 as the precipitant. One form diffracted up to 2.4 A resolution with synchrotron radiation at 100 K, but suffered serious X-ray damage. It belongs to the space group P2(1)2(1)2(1) with unit-cell parameters of a = 47.9, b = 94.9, c = 125.2 A. The other form is a thin plate, but the data sets were successfully collected up to 2.4 A resolution by use of synchrotron radiation at 100 K. The crystals belong to the space group P2(1) with unit-cell parameters of a = 57.4, b = 51.2, c = 101.1 A, and beta = 97.9 degrees, and contain one molecule per asymmetric unit with a VM value of 2.05 A(3)/Da.


Subject(s)
Antigens, CD , Antigens, Differentiation/chemistry , Antigens, Differentiation/metabolism , Gangliosides/chemistry , Gangliosides/metabolism , NAD+ Nucleosidase/chemistry , NAD+ Nucleosidase/metabolism , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Antigens, Differentiation/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Crystallization , Maltose-Binding Proteins , NAD+ Nucleosidase/genetics , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , X-Ray Diffraction
14.
Structure ; 8(2): 197-208, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10673435

ABSTRACT

BACKGROUND: The 20 aminoacyl-tRNA synthetases are divided into two classes, I and II. The 10 class I synthetases are considered to have in common the catalytic domain structure based on the Rossmann fold, which is totally different from the class II catalytic domain structure. The class I synthetases are further divided into three subclasses, a, b and c, according to sequence homology. No conserved structural features for tRNA recognition by class I synthetases have been established. RESULTS: We determined the crystal structure of the class Ia methionyl-tRNA synthetase (MetRS) at 2.0 A resolution, using MetRS from an extreme thermophile, Thermus thermophilus HB8. The T. thermophilus MetRS structure is in full agreement with the biochemical and genetic data from Escherichia coli MetRS. The conserved 'anticodon-binding' residues are spatially clustered on an alpha-helix-bundle domain. The Rossmann-fold and anticodon-binding domains are connected by a beta-alpha-alpha-beta-alpha topology ('SC fold') domain that contains the class I specific KMSKS motif. CONCLUSIONS: The alpha-helix-bundle domain identified in the MetRS structure is the signature of the class Ia enzymes, as it was also identified in the class Ia structures of the isoleucyl- and arginyl-tRNA synthetases. The beta-alpha-alpha-beta-alpha topology domain, which can now be identified in all known structures of the class Ia and Ib synthetases, is likely to dock with the inner side of the L-shaped tRNA, thereby positioning the anticodon stem.


Subject(s)
Methionine-tRNA Ligase/chemistry , RNA-Binding Proteins/chemistry , Thermus thermophilus/chemistry , Anticodon , Catalytic Domain , Crystallography, X-Ray , Methionine-tRNA Ligase/metabolism , Models, Molecular , Protein Conformation , Protein Folding , RNA-Binding Proteins/metabolism
15.
J Mol Biol ; 294(5): 1287-97, 1999 Dec 17.
Article in English | MEDLINE | ID: mdl-10600385

ABSTRACT

The 3D structure of monomeric C-truncated Escherichia coli methionyl-tRNA synthetase, a class 1 aminoacyl-tRNA synthetase, has been solved at 2.0 A resolution. Remarkably, the polypeptide connecting the two halves of the Rossmann fold exposes two identical knuckles related by a 2-fold axis but with zinc in the distal knuckle only. Examination of available MetRS orthologs reveals four classes according to the number and zinc content of the putative knuckles. Extreme cases are exemplified by the MetRS of eucaryotic or archaeal origin, where two knuckles and two metal ions are expected, and by the mitochondrial enzymes, which are predicted to have one knuckle without metal ion.


Subject(s)
Escherichia coli/enzymology , Methionine-tRNA Ligase/chemistry , Methionine-tRNA Ligase/classification , Amino Acid Sequence , Animals , Anticodon/metabolism , Binding Sites , Catalytic Domain , Crystallization , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Sequence Alignment , Static Electricity , Zinc/metabolism
16.
Nature ; 398(6728): 579-85, 1999 Apr 15.
Article in English | MEDLINE | ID: mdl-10217141

ABSTRACT

The Sex-lethal (Sxl) protein of Drosophila melanogaster regulates alternative splicing of the transformer (tra) messenger RNA precursor by binding to the tra polypyrimidine tract during the sex-determination process. The crystal structure has now been determined at 2.6 A resolution of the complex formed between two tandemly arranged RNA-binding domains of the Sxl protein and a 12-nucleotide, single-stranded RNA derived from the tra polypyrimidine tract. The two RNA-binding domains have their beta-sheet platforms facing each other to form a V-shaped cleft. The RNA is characteristically extended and bound in this cleft, where the UGUUUUUUU sequence is specifically recognized by the protein. This structure offers the first insight, to our knowledge, into how a protein binds specifically to a cognate RNA without any intramolecular base-pairing.


Subject(s)
Drosophila Proteins , Nuclear Proteins/genetics , RNA Precursors/chemistry , RNA-Binding Proteins/chemistry , Amino Acid Sequence , Animals , Base Pairing , Crystallography, X-Ray , Drosophila melanogaster , Escherichia coli , Insect Hormones/chemistry , Insect Hormones/metabolism , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Nuclear Proteins/chemistry , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Protein Structure, Secondary , Pyrimidines/chemistry , RNA Precursors/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment
17.
Eur J Biochem ; 261(2): 354-60, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10215844

ABSTRACT

We previously elucidated the major determinant set for Escherichia coli tRNAGlu identity (U34, U35, C36, A37, G1*C72, U2*A71, U11*A24, U13*G22**Alpha46, and Delta47) and showed that the set is sufficient to switch the identity of tRNAGln to Glu [Sekine, S., Nureki, O., Sakamoto, K., Niimi, T., Tateno, M., Go, M., Kohno, T., Brisson, A., Lapointe, J. & Yokoyama, S. (1996) J. Mol. Biol. 256, 685-700]. In the present study, we attempted to switch the identity of tRNAAsp, which has a sequence similar to that of tRNAGlu, and consequently possesses many nucleotide residues corresponding to the Glu identity determinants (U35, C36, A37, G1*C72, and U11*A24). A simple transplantation of the rest of the major determinants (U34, U2*A71, U13*G22**Alpha46, and Delta47) to the framework of tRNAAsp did not result in a sufficient switch of the tRNAAsp identity to Glu. To confer an optimal glutamate accepting activity to tRNAAsp, two other elements, C4*G69 in the middle of the acceptor stem and C12*G23**C9 in the augmented D helix, were required. Consistently, the two base pairs, C4*G69 and C12*G23, in tRNAGlu had been shown to exist in the interface with glutamyl-tRNA synthetase (GluRS) by phosphate-group footprinting. We also found the two elements in the framework of tRNAGln, and determined that their contributions successfully changed the identity of tRNAGln to Glu in the previous study. By the identity-determinant set (C4*G69 and C12*G23**C9 in addition to U34, U35, C36, A37, G1*C72, U2*A71, U11*A24, U13*G22**Alpha46, and Delta47) the activity of GluRS was optimized and efficient discrimination from the noncognate tRNAs was achieved.


Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Escherichia coli/enzymology , RNA, Transfer, Asp/chemistry , RNA, Transfer, Glu/chemistry , Acylation , Cloning, Molecular , Kinetics , Models, Molecular , Mutation , Nucleic Acid Conformation , RNA, Transfer, Asp/genetics , RNA, Transfer, Glu/genetics , Substrate Specificity
18.
Science ; 280(5363): 578-82, 1998 Apr 24.
Article in English | MEDLINE | ID: mdl-9554847

ABSTRACT

High-fidelity transfers of genetic information in the central dogma can be achieved by a reaction called editing. The crystal structure of an enzyme with editing activity in translation is presented here at 2.5 angstroms resolution. The enzyme, isoleucyl-transfer RNA synthetase, activates not only the cognate substrate L-isoleucine but also the minimally distinct L-valine in the first, aminoacylation step. Then, in a second, "editing" step, the synthetase itself rapidly hydrolyzes only the valylated products. For this two-step substrate selection, a "double-sieve" mechanism has already been proposed. The present crystal structures of the synthetase in complexes with L-isoleucine and L-valine demonstrate that the first sieve is on the aminoacylation domain containing the Rossmann fold, whereas the second, editing sieve exists on a globular beta-barrel domain that protrudes from the aminoacylation domain.


Subject(s)
Isoleucine-tRNA Ligase/chemistry , Isoleucine/metabolism , Valine/metabolism , Adenosine Monophosphate , Binding Sites , Crystallography, X-Ray , Escherichia coli/enzymology , Hydrogen Bonding , Hydrolysis , Isoleucine-tRNA Ligase/metabolism , Models, Chemical , Models, Molecular , Molecular Sequence Data , Mutagenesis , Protein Conformation , Protein Folding , Protein Structure, Secondary , RNA, Transfer, Ile/metabolism , Substrate Specificity , Thermus thermophilus/enzymology , Transfer RNA Aminoacylation
19.
J Mol Biol ; 256(4): 685-700, 1996 Mar 08.
Article in English | MEDLINE | ID: mdl-8642591

ABSTRACT

By a kinetic analysis of 59 variant transcripts of Escherichia coli tRNA(Glu) with glutamyl-tRNA synthetase (GluRS), the U11.A24 base-pair, the U13.G22..A46 base-triple, and the lack of residue 47 (delta47) were found to serve as major determinants for tRNA(Glu) identity. This is the first system for which major identity determinants are reported to be clustered in the "augmented D helix", consisting of the D stem with some neighboring residues and the variable loop. Other identity determinants are U34, U35, C36 and A37 in the anticodon loop, and G1.C72 and U2.A71 in the acceptor stem. Phosphate-group protection by GluRS from ethylnitrosourea was observed most strongly for the minor groove side of D-stem helix, indicating that GluRs tightly binds to the D stem for recognition, on the minor groove side, of the potent identity-determinant groups of the U11.A24 and U13.G22 base-pairs. A46 is not involved in direct recognition by GluRS; the U13.G22..A46 base-triple is required probably for formation of the structural features that are recognized by GluRS. In this context, the essential role of characteristic delta47 in tRNA(Glu) identity may be to maintain the U13.G22..A46 base-triple.


Subject(s)
Escherichia coli/chemistry , Nucleic Acid Conformation , RNA, Transfer, Glu/chemistry , Anticodon/genetics , Base Composition , Base Sequence , Cloning, Molecular , Codon/genetics , Electrophoresis, Polyacrylamide Gel , Ethylnitrosourea/metabolism , Glutamate-tRNA Ligase/metabolism , Hydrogen Bonding , Kinetics , Models, Molecular , Molecular Sequence Data , Oligoribonucleotides/chemistry , Protein Binding , RNA, Transfer, Gln/chemistry , RNA, Transfer, Gln/metabolism , RNA, Transfer, Glu/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism
20.
FEBS Lett ; 377(1): 77-81, 1995 Dec 11.
Article in English | MEDLINE | ID: mdl-8543024

ABSTRACT

A docking model of glutamyl-tRNA synthetase (GluRS) and tRNAGlu was constructed, on the basis of the distinguished similarity between the X-ray crystallographic three-dimensional structures of the N-terminal halves of the Thermus thermophilus GluRS in the free state and the Escherichia coli glutaminyl-tRNA synthetase in a complex with tRNAGln. The modeled structure is energetically favorable and is also well consistent with the results of site-directed mutagenesis studies. The model indicates that the GluRS-specific insertions 2 and 3 fit and bind to the acceptor stem and the D arm, respectively, of the cognate tRNA without affecting other contacts. In particular, insertion 3 strongly interacts with the two D-stem base pairs that are essential for the tRNA-GluRS recognition.


Subject(s)
Glutamate-tRNA Ligase/chemistry , Models, Molecular , RNA, Transfer, Glu/chemistry , Anticodon , Binding Sites , Crystallography, X-Ray , Escherichia coli/enzymology , Glutamate-tRNA Ligase/genetics , Glutamate-tRNA Ligase/metabolism , Mutagenesis, Site-Directed , Nucleic Acid Conformation , RNA, Transfer, Glu/genetics , RNA, Transfer, Glu/metabolism , Thermodynamics , Thermus thermophilus/enzymology
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