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At least 289 records · Page 16Linked to original sources
PubMed2000

tRNA aminoacylation by arginyl-tRNA synthetase: induced conformations during substrates binding.

The 2.2 A crystal structure of a ternary complex formed by yeast arginyl-tRNA synthetase and its cognate tRNA(Arg) in the presence of the L-arginine substrate highlights new atomic features used for specific substrate recognition. This first example of an active complex formed by a class Ia aminoacyl-tRNA synthetase and its natural cognate tRNA illustrates additional strategies used for specific tRNA selection. The enzyme specifically recognizes the D-loop and the anticodon of the tRNA, and the mutually induced fit produces a conformation of the anticodon loop never seen before. Moreover, the anticodon binding triggers conformational changes in the catalytic center of the protein. The comparison with the 2.9 A structure of a binary complex formed by yeast arginyl-tRNA synthetase and tRNA(Arg) reveals that L-arginine binding controls the correct positioning of the CCA end of the tRNA(Arg). Important structural changes induced by substrate binding are observed in the enzyme. Several key residues of the active site play multiple roles in the catalytic pathway and thus highlight the structural dynamics of the aminoacylation reaction.

Anticodon↗
PubMed1993

The long extra arms of human tRNA((Ser)Sec) and tRNA(Ser) function as major identify elements for serylation in an orientation-dependent, but not sequence-specific manner.

Selenocysteine tRNA [tRNA((Ser)Sec)] is charged with serine by the same seryl-tRNA synthetase (SerRS) as the canonical serine tRNAs. Using site-directed mutagenesis, we have introduced a series of mutations into human tRNA((Ser)Sec) and tRNA(Ser) in order to study the identity elements of tRNA((Ser)Sec) for serylation and the effect of the orientation of the extra arm. Our results show that the long extra arm is one of the major identity elements for both tRNA(Ser) and tRNA((Ser)Sec) and gel retardation assays reveal that it appears to be a prerequisite for binding to the cognate synthetase. The long extra arm functions in an orientation-dependent, but not in a sequence-specific manner. The discriminator base G73 is another important identity element of tRNA((Ser)Sec), whereas the T- and D-arms play a minor role for the serylation efficiency.

Base Sequence↗
PubMed2004

Two distinct domains of the beta subunit of Aquifex aeolicus leucyl-tRNA synthetase are involved in tRNA binding as revealed by a three-hybrid selection.

The Aquifex aeolicus alphabeta-LeuRS is the only known heterodimeric class Ia aminoacyl-tRNA synthetase. In this study, we investigated the function of the beta subunit which is believed to bind tRNA(Leu). A yeast three-hybrid system was constructed on the basis of the interaction of the beta subunit with its cognate tRNA(Leu). Then, seven mutated beta subunits exhibiting impaired tRNA binding capacities were selected out from a randomly mutated library. Two mutations were identified in the class Ia-helix-bundle-domain, which might interact with the D-hairpin of the tRNA analogous to other class Ia tRNA:synthetases complexes. The five other mutations were found in the LeuRS-specific C-terminal domain of which the folding is still unknown. tRNA affinity measurements and kinetic analyses performed on the isolated beta subunits and on the co-expressed alphabeta-heterodimers showed for all the mutants an effect in tRNA affinity in the ground state. In addition, an effect on the transition state of the aminoacylation reaction was observed for a 21-residues deletion mutant of the C-terminal end. These results show that the genetic approach of the three hybrid system is widely applicable and is a powerful tool for the investigation of tRNA:synthetase interactions.

Amino Acid Sequence↗
PubMed1982

Conformational activation of aminoacyl-tRNA synthetases upon binding of tRNA. A facet of a multi-step adaptation process leading to the optimal biological activity.

The activation of the catalytic center of aminoacyl-tRNA synthetases upon binding of the tRNA, previously reported in the case of yeast phenylalanyl-tRNA and valyl-tRNA synthetases [Renaud et al., (1981) Proc. Natl Acad. Sci. USA, 78, 1606-1608] has been investigated in other systems. It is shown that this property is encountered not only in cognate systems (phenylalanyl, valyl and arginyl) but also in the non-cognate systems which are particularly efficient in misaminoacylation reactions. The arginyl system, the peculiarity of which is to form the aminoacyladenylate only in the presence of the cognate tRNA, is shown to be a border-line case of this general process of catalytic center activation. In the case of the phenylalanyl system, the crucial role of the wybutine residue (adjacent to the anticodon) in the activation of phenylalanyl-tRNA synthetase by the tRNA core has been analysed by comparison with native or modified non-cognate tRNAs (tRNATyr, tRNAArg). It is proposed that upon complex formation between a tRNA and its cognate aminoacyl-tRNA synthetase, a multistep adaptation process takes place in order to promote the optimal rate for the aminoacylation reaction, thus contributing to the specificity of this reaction.

Adaptation, Biological↗
PubMed1990

High-molecular-weight forms of aminoacyl-tRNA synthetases and tRNA modification enzymes in Escherichia coli.

The presence of high-molecular-weight complexes of aminoacyl-tRNA synthetases in Escherichia coli has been reported (C. L. Harris, J. Bacteriol. 169:2718-2723, 1987). In the current study, Bio-Gel A-5M gel chromatography of 105,000 x g supernatant preparations from E. coli Q13 indicated high molecular weights for both tRNA methylase (300,000) and tRNA sulfurtransferase (450,000). These tRNA modification enzymes did not appear to exist in the same multienzymic complex. On the other hand, 4-thiouridine sulfurtransferase eluted with aminoacyl-tRNA synthetase activity on Bio-Gel A-5M, and both of these activities were cosedimented after further centrifugation of cell supernatants at 160,000 x g for 18 h. Despite this evidence for association of the sulfurtransferase with the synthetase complex, isoleucyl-tRNA synthetase and tRNA sulfurtransferase were totally resolved from each other by DEAE-Sephacel chromatography. Subsequent gel chromatography showed little change in their elution positions on agarose. Hence, either nonspecific aggregation occurred here, or the modification enzymes studied are not members of the aminoacyl-tRNA synthetase complex in E. coli. These findings do suggest that some bacterial tRNA modification enzymes are present in multiprotein complexes of high molecular weight.

Amino Acyl-tRNA Synthetases↗
PubMed2003

Mechanism of molecular interactions for tRNA(Val) recognition by valyl-tRNA synthetase.

The molecular interactions between valyl-tRNA synthetase (ValRS) and tRNA(Val), with the C34-A35-C36 anticodon, from Thermus thermophilus were studied by crystallographic analysis and structure-based mutagenesis. In the ValRS-bound structure of tRNA(Val), the successive A35-C36 residues (the major identity elements) of tRNA(Val) are base-stacked upon each other, and fit into a pocket on the alpha-helix bundle domain of ValRS. Hydrogen bonds are formed between ValRS and A35-C36 of tRNA(Val) in a base-specific manner. The C-terminal coiled-coil domain of ValRS interacts electrostatically with A20 and hydrophobically with the G19*C56 tertiary base pair. The loss of these interactions by the deletion of the coiled-coil domain of ValRS increased the K(M) value for tRNA(Val) 28-fold and decreased the k(cat) value 19-fold in the aminoacylation. The tRNA(Val) K(M) and k(cat) values were increased 21-fold and decreased 32-fold, respectively, by the disruption of the G18*U55 and G19*C56 tertiary base pairs, which associate the D- and T-loops for the formation of the L-shaped tRNA structure. Therefore, the coiled-coil domain of ValRS is likely to stabilize the L-shaped tRNA structure during the aminoacylation reaction.

Anticodon↗
PubMed1990

Glutamyl-tRNA synthetases of Bacillus subtilis 168T and of Bacillus stearothermophilus. Cloning and sequencing of the gltX genes and comparison with other aminoacyl-tRNA synthetases.

The glutamyl-tRNA synthetase (GluRS) of Bacillus subtilis 168T aminoacylates with glutamate its homologous tRNA(Glu) and tRNA(Gln) in vivo and Escherichia coli tRNA(1Gln) in vitro (Lapointe, J., Duplain, L., and Proulx, M. (1986) J. Bacteriol. 165, 88-93). The gltX gene encoding this enzyme was cloned and sequenced. It encodes a protein of 483 amino acids with a Mr of 55,671. Alignment of the amino acid sequences of four bacterial GluRSs (from B. subtilis, Bacillus stearothermophilus, E. coli, and Rhizobium meliloti) gives 20% identity and reveals the presence of several short highly conserved motifs in the first two thirds of these proteins. Conserved motifs are found at corresponding positions in several other aminoacyl-tRNA synthetases. The only sequence similarity between the GluRSs of these Bacillus species and the E. coli glutaminyl-tRNA synthetase (GlnRS), which has no counterpart in the E. coli GluRS, is in a segment of 30 amino acids in the last third of these synthetases. In the three-dimensional structure of the E. coli tRNA(Gln).GlnRS.ATP complex, this conserved peptide is near the anticodon of tRNA(Gln) (Rould, M. A., Perona, J. J., Söll, D., and Steitz, T. A. (1989) Science 246, 1135-1142), suggesting that this region is involved in the specific interactions between these enzymes and the anticodon regions of their tRNA substrates.

Amino Acid Sequence↗
PubMedDate not supplied

[Temperature-dependent structural changes in complexes of tRNA(adenine-1)-methyltransferase from Thermus thermophilus HB8 and tRNA studied by optical and electron microscope methods].

The complexation of tRNA (adenine-1-)-methyltransferase from Thermus thermophilus HB8 (E.C.2.1.1.36) with Escherichia coli tRNA(Phe) and yeast tRNA1(Val) was investigated in a temperature range from 20 to 90 degrees C. The quantity of methylase subunits bounded with tRNA and the association constant (Ka) were determined by means of fluorescence quenching of the enzyme tryptophane residues by tRNA molecules. The number of enzyme subunits bounded with one tRNA molecule at temperatures 20-70 degrees C is equal to 8 +/- 2. The Ka values increase from (2 divided by 3).10(7) at 20 degrees C up to 8.5.10(7) M-1 at 70 degrees C. The temperature increase from 70 to 90 degrees C causes a decrease in the enzyme specific activity and in Ka values. In the temperature range from 75 to 90 degrees C a cooperative transition of methylase macromolecules into associates was observed. This association is accompanied by an increase of UV-light scattering and of fluorescence polarization coefficient of methylase tryptophane residues. In the absence of tRNA the size of enzyme associates (d) is evaluated to be more than 320 nm (d greater than or equal to lambda-320 nm), in the presence of tRNA-less than 320 nm (d much less than lambda-320 nm). An electron microscopic investigation of methylase and its complexes with tRNA at 20 degrees C revealed disk-like particles with a diameter and height of 8-11 nm and 4-5 nm, respectively. These disk-like methylase preparations dialized against distilled water form flexible polymeric rods with a diameter of 10-12 nm and the length of about several hundreds nm. During complexation of methylase with tRNA, in the same conditions as the dializes was carried out, large associates were not revealed.

Escherichia coli↗
PubMedDate not supplied

[Stability of phosphodiester tRNA bonds lacking minor nucleosides, in aminoacyl tRNA-synthetase complexes].

The stability of phosphodiester bonds in tRNA(Trp) (E. coli, bovine) and tRNA(Phe) (yeast) synthesized in vitro was studied in the complexes with cognate aminoacyl-tRNA synthetases. In contrast to E. coli tRNA(Gln) where synthetase-induced cleavage has been reported, we have failed to observe this effect with tRNA(Trp) and tRNA(Phe). Consequently, the weakness of sugar-phosphate backbone of tRNA-transcripts in the complexes is not a common feature for all tRNA-synthetase pairs. The cleavage mechanism in the case of tRNA(Gln) remains obscure.

Amino Acyl-tRNA Synthetases↗
PubMed1993

Saccharomyces cerevisiae cytoplasmic tyrosyl-tRNA synthetase gene. Isolation by complementation of a mutant Escherichia coli suppressor tRNA defective in aminoacylation and sequence analysis.

Exploiting differences in tRNA recognition between prokaryotic and eukaryotic tyrosyl-tRNA synthetases (TyrRSs), we have isolated the gene for the cytoplasmic TyrRS of Saccharomyces cerevisiae by functional complementation in Escherichia coli of a mutant E. coli tRNA. The tRNA, derived from the E. coli initiator tRNA with changes to allow suppression of amber termination codons, is poorly aminoacylated in E. coli and hence, is only a weak amber suppressor. The same tRNA functions as a good suppressor in S. cerevisiae and is aminoacylated with tyrosine by yeast extracts. We expressed a yeast cDNA library in an E. coli strain carrying the mutant tRNA gene and several genes with amber mutations. cDNA clones were isolated which increased suppression and levels of aminoacylation of the mutant tRNA. Characterization of the gene identified a methionine-initiated open reading frame encoding a protein of 394 amino acids. Expression of this protein in E. coli demonstrated that tyrosine was incorporated during suppression and that yeast cytoplasmic TyrRS activity was produced. Yeast cytoplasmic TyrRS has sequences typical of class I aminoacyl-tRNA synthetases, but only weak overall sequence similarity to the corresponding eubacterial and mitochondrial TyrRSs. However, many of the residues known to line the tyrosyl-adenylate-binding pocket of the Bacillus stearothermophilus enzyme can be aligned in the yeast sequence. These include the aspartic acid and tyrosine residues thought to contact the tyrosine side chain to provide substrate specificity.

Amino Acid Sequence↗
PubMed2002

Histidyl-tRNA synthetase and asparaginyl-tRNA synthetase, autoantigens in myositis, activate chemokine receptors on T lymphocytes and immature dendritic cells.

Autoantibodies to histidyl-tRNA synthetase (HisRS) or to alanyl-, asparaginyl-, glycyl-, isoleucyl-, or threonyl-tRNA synthetase occur in approximately 25% of patients with polymyositis or dermatomyositis. We tested the ability of several aminoacyl-tRNA synthetases to induce leukocyte migration. HisRS induced CD4(+) and CD8(+) lymphocytes, interleukin (IL)-2-activated monocytes, and immature dendritic cells (iDCs) to migrate, but not neutrophils, mature DCs, or unstimulated monocytes. An NH(2)-terminal domain, 1-48 HisRS, was chemotactic for lymphocytes and activated monocytes, whereas a deletion mutant, HisRS-M, was inactive. HisRS selectively activated CC chemokine receptor (CCR)5-transfected HEK-293 cells, inducing migration by interacting with extracellular domain three. Furthermore, monoclonal anti-CCR5 blocked HisRS-induced chemotaxis and conversely, HisRS blocked anti-CCR5 binding. Asparaginyl-tRNA synthetase induced migration of lymphocytes, activated monocytes, iDCs, and CCR3-transfected HEK-293 cells. Seryl-tRNA synthetase induced migration of CCR3-transfected cells but not iDCs. Nonautoantigenic aspartyl-tRNA and lysyl-tRNA synthetases were not chemotactic. Thus, autoantigenic aminoacyl-tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses. Therefore, the selection of a self-molecule as a target for an autoantibody response may be a consequence of the proinflammatory properties of the molecule itself.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Structure of the yeast valyl-tRNA synthetase gene (VASI) and the homology of its translated amino acid sequence with Escherichia coli isoleucyl-tRNA synthetase.

The VASI gene encoding the valyl-tRNA synthetase from yeast was isolated and sequenced. The gene-derived amino acid sequence of yeast valyl-tRNA synthetase was found to be 23% homologous to the Escherichia coli isoleucyl-tRNA synthetase. This is the highest level of homology reported so far between two distinct aminoacyl-tRNA synthetases and is indicative of an evolutionary relationship between these two molecules. Within these homologous sequences, two functional regions could be recognized: the HIGH region which forms part of the binding site of ATP and the KMSKS region which is recognized as the consensus sequence for the binding of the 3'-end of tRNA (Hountondji, C., Dessen, Ph., and Blanquet, S. (1986) Biochemie (Paris) 68, 1071-1078). Secondary structure predictions as well as the presence of both HIGH and KMSKS regions, delineating the nucleotide-binding domain and the COOH-terminal helical domain in aminoacyl-tRNA synthetases of known three-dimensional structure, suggest that the yeast valyl-tRNA synthetase polypeptide chain can be folded into three domains: an NH2-terminal alpha-helical region followed by a nucleotide-binding topology and a COOH-terminal domain composed of alpha-helices which probably carries major sites in tRNA binding.

Adenosine Triphosphate↗