[Encapsidation of cellular tRNA by HIV-1]. / Détournement d'ARNt cellulaire par le VIH1.

Human immunodeficiency virus type 1, HIV1, is a retrovirus. Its genome is made of two single-stranded RNA molecules. Reverse transcriptase encoded by HIV1 uses tRNA3 Lys of the host cell to start the reverse transcription of its RNA genome into proviral DNA. During retroviral budding, this tRNA is packaged into the viral particles. Molecular mechanisms involved in viral hijacking of molecules of the host cell are described.

The appended C-domain of human methionyl-tRNA synthetase has a tRNA-sequestering function.

An ancillary RNA-binding domain is appended to the C-terminus of human methionyl-tRNA synthetase. It comprises a helix-turn-helix (HTH) motif related to the repeated units of the linker region of bifunctional glutamyl-prolyl-tRNA synthetase, and a specific C-terminal KGKKKK lysine-rich cluster (LRC). Here we show by gel retardation and tRNA aminoacylation experiments that these two regions are important for tRNA binding. However, the two pieces of this bipartite RNA-binding domain are functionally distinct. Analysis of MetRS mutant enzymes revealed that the HTH motif is more specifically endowed with a tRNA-sequestering activity and confers on MetRS a rate-limiting dissociation of aminoacylated tRNA. Elongation factor EF-1alpha enhanced the turnover in the aminoacylation reaction. In contrast, the LRC region is most probably involved in accelerating the association step of deacylated tRNA. These two nonredundant RNA-binding motifs strengthen tRNA binding by the synthetase. The native form of MetRS, containing the C-terminal RNA-binding domain, behaves as a processive enzyme; release of the reaction product is not spontaneous, but may be synchronized with the subsequent step of the tRNA cycle through EF-1alpha-assisted dissociation of Met-tRNA(Met). Therefore, the eukaryotic-specific C-domain of human MetRS may have a dual function. It may ensure an efficient capture of tRNA(Met) under conditions of suboptimal deacylated tRNA concentration prevailing in vivo, and may instigate direct transfer of aminoacylated tRNA from the synthetase to elongation factor EF-1alpha.

The EMAPII cytokine is released from the mammalian multisynthetase complex after cleavage of its p43/proEMAPII component.

Endothelial-monocyte-activating polypeptide II (EMAPII) is an inflammatory cytokine released under apoptotic conditions. Its proEMAPII precursor proved to be identical to the auxiliary p43 component of the aminoacyl-tRNA synthetase complex. We show here that the EMAPII domain of p43 is released readily from the complex after in vitro digestion with caspase 7 and is able to induce migration of human mononuclear phagocytes. The N terminus of in vitro-processed EMAPII coincides exactly with that of the mature cytokine isolated from conditioned medium of fibrosarcoma cells. We also show that p43/proEMAPII has a strong tRNA binding capacity (K(D) = 0.2 microm) as compared with its isolated N or C domains (7.5 microm and 40 microm, respectively). The potent general RNA binding capacity ascribed to p43/proEMAPII is lost upon the release of the EMAPII domain. This suggests that after onset of apoptosis, the first consequence of the cleavage of p43 is to limit the availability of tRNA for aminoacyl-tRNA synthetases associated within the complex. Translation arrest is accompanied by the release of the EMAPII cytokine that plays a role in the engulfment of apoptotic cells by attracting phagocytes. As a consequence, p43 compares well with a molecular fuse that triggers the irreversible cell growth/cell death transition induced under apoptotic conditions.

Structure of the EMAPII domain of human aminoacyl-tRNA synthetase complex reveals evolutionary dimer mimicry.

The EMAPII (endothelial monocyte-activating polypeptide II) domain is a tRNA-binding domain associated with several aminoacyl-tRNA synthetases, which becomes an independent domain with inflammatory cytokine activity upon apoptotic cleavage from the p43 component of the multisynthetase complex. It comprises a domain that is highly homologous to bacterial tRNA-binding proteins (Trbp), followed by an extra domain without homology to known proteins. Trbps, which may represent ancient tRNA chaperones, form dimers and bind one tRNA per dimer. In contrast, EMAPII domains are monomers. Here we report the crystal structure at 1.14 Angstroms of human EMAPII. The structure reveals that the Trbp-like domain, which forms an oligonucleotide-binding (OB) fold, is related by degenerate 2-fold symmetry to the extra-domain. The pseudo-axis coincides with the dyad axis of bacterial TtCsaA, a Trbp whose structure was solved recently. The interdomain interface in EMAPII mimics the intersubunit interface in TtCsaA, and may thus generate a novel OB-fold-based tRNA-binding site. The low sequence homology between the extra domain of EMAPII and either its own OB fold or that of Trbps suggests that dimer mimicry originated from convergent evolution rather than gene duplication.

Macromolecular assemblage of aminoacyl-tRNA synthetases: quantitative analysis of protein-protein interactions and mechanism of complex assembly.

The structure of the mammalian multi-synthetase complex was investigated in vitro using qualitative and quantitative approaches. This macromolecular assemblage comprises the bifunctional glutamyl-prolyl-tRNA synthetase, the seven monospecific isoleucyl, leucyl, methionyl, glutaminyl, lysyl, arginyl and aspartyl-tRNA synthetases, and the three auxiliary p43, p38 and p18 proteins. The scaffold p38 protein was expressed in Escherichia coli and purified to homogeneity as a His-tagged protein. The different components of the complex were shown to associate in vitro with p38 immobilized on Ni(2+)-coated plates. Interactions between peripheral enzymes and p38 are referred to as central interactions, as opposed to lateral interactions between peripheral enzymes. Kinetic parameters of the interactions were determined by the means of a biosensor-based approach. The two dimeric proteins LysRS and AspRS were found to tightly bind to p38, with a K(d) value of 0.3 and 4.7 nM, respectively. These interactions involved the catalytic core of the enzymes. By contrast, binding of ArgRS or GlnRS to p38 was much weaker (>5 microM). ArgRS and p43, two peripheral components, were shown to interact with moderate affinity (K(d)=93 nM). Since all the components of the complex are tightly associated within this particle, lateral interactions were believed to contribute to the stabilization of this assemblage. Using an in vitro binding assay, concomitant association of several components of the complex on immobilized p38 could be demonstrated, and revealed the involvement of synergistic effects for association of weakly interacting proteins. Taking into account the possible synergy between central and lateral contributions, a sub-complex containing p38, p43, ArgRS and GlnRS was reconstituted in vitro. These data provide compelling evidence for an ordered and concerted mechanism of complex assembly.

A recurrent general RNA binding domain appended to plant methionyl-tRNA synthetase acts as a cis-acting cofactor for aminoacylation.

The cDNA encoding rice methionyl-tRNA synthetase was isolated. The protein exhibited a C-terminal polypeptide appended to a classical MetRS domain. This supplementary domain is related to endothelial monocyte activating polypeptide II (EMAPII), a cytokine produced in mammals after cleavage of p43, a component of the multisynthetase complex. It is also related to Arc1p and Trbp111, two tRNA binding proteins. We expressed rice MetRS and a derivative with a deletion of its EMAPII-like domain. Band-shift analysis showed that this extra-domain provides MetRS with non-specific tRNA binding properties. The EMAPII-like domain contributed a 10-fold decrease in K:(M) for tRNA in the aminoacylation reaction catalyzed by the native enzyme, as compared with the C-terminally truncated MetRS. Consequently, the EMAPII domain provides MetRS with a better catalytic efficiency at the free tRNA concentration prevailing in vivo. This domain binds the acceptor minihelix of tRNA(Met) and facilitates its aminoacylation. These results suggest that the EMAPII module could be a relic of an ancient tRNA binding domain that was incorporated into primordial synthetases for aminoacylation of RNA minihelices taken as the ancestor of modern tRNA.

The tRNA-dependent activation of arginine by arginyl-tRNA synthetase requires inter-domain communication.

The tRNA-dependent amino acid activation catalyzed by mammalian arginyl-tRNA synthetase has been characterized. A conditional lethal mutant of Chinese hamster ovary cells that exhibits reduced arginyl-tRNA synthetase activity (Arg-1), and two of its derived revertants (Arg-1R4 and Arg-1R5) were analyzed at the structural and functional levels. A single nucleotide change, resulting in a Cys to Tyr substitution at position 599 of arginyl-tRNA synthetase, is responsible for the defective phenotype of the thermosensitive and arginine hyper-auxotroph Arg-1 cell line. The two revertants have a single additional mutation resulting in a Met222 to Ile change for Arg-1R4 or a Tyr506 to Ser change for Arg-1R5. The corresponding mutant enzymes were expressed in yeast and purified. The Cys599 to Tyr mutation affects both the thermal stability of arginyl-tRNA synthetase and the kinetic parameters for arginine in the ATP-PP(i) exchange and tRNA aminoacylation reactions. This mutation is located underneath the floor of the Rossmann fold catalytic domain characteristic of class 1 aminoacyl-tRNA synthetases, near the end of a long helix belonging to the alpha-helix bundle C-terminal domain distinctive of class 1a synthetases. For the Met222 to Ile revertant, there is very little effect of the mutation on the interaction of arginyl-tRNA synthetase with either of its substrates. However, this mutation increases the thermal stability of arginyl-tRNA synthetase, thereby leading to reversion of the thermosensitive phenotype by increasing the steady-state level of the enzyme in vivo. In contrast, for the Arg-1R5 cell line, reversion of the phenotype is due to an increased catalytic efficiency of the C599Y/Y506S double mutant as compared to the initial C599Y enzyme. In light of the location of the mutations in the 3D structure of the enzyme modeled using the crystal structure of the closely related yeast arginyl-tRNA synthetase, the kinetic analysis of these mutants suggests that the obligatory tRNA-induced activation of the catalytic site of arginyl-tRNA synthetase involves interdomain signal transduction via the long helices that build the tRNA-binding domain of the enzyme and link the site of interaction of the anticodon domain of tRNA to the floor of the active site.

A gene fusion event in the evolution of aminoacyl-tRNA synthetases.

The genes of glutamyl- and prolyl-tRNA synthetases (GluRS and ProRS) are organized differently in the three kingdoms of the tree of life. In bacteria and archaea, distinct genes encode the two proteins. In several organisms from the eukaryotic phylum of coelomate metazoans, the two polypeptides are carried by a single polypeptide chain to form a bifunctional protein. The linker region is made of imperfectly repeated units also recovered as singular or plural elements connected as N-terminal or C-terminal polypeptide extensions in various eukaryotic aminoacyl-tRNA synthetases. Phylogenetic analysis points to the monophyletic origin of this polypeptide motif appended to six different members of the synthetase family, belonging to either of the two classes of aminoacyl-tRNA synthetases. In particular, the monospecific GluRS and ProRS from Caenorhabditis elegans, an acoelomate metazoan, exhibit this recurrent motif as a C-terminal or N-terminal appendage, respectively. Our analysis of the extant motifs suggests a possible series of events responsible for a gene fusion that gave rise to the bifunctional glutamyl-prolyl-tRNA synthetase through recombination between genomic sequences encoding the repeated units.

A recurrent RNA-binding domain is appended to eukaryotic aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases of higher eukaryotes possess polypeptide extensions in contrast to their prokaryotic counterparts. These extra domains of poorly understood function are believed to be involved in protein-protein or protein-RNA interactions. Here we showed by gel retardation and filter binding experiments that the repeated units that build the linker region of the bifunctional glutamyl-prolyl-tRNA synthetase had a general RNA-binding capacity. The solution structure of one of these repeated motifs was also solved by NMR spectroscopy. One repeat is built around an antiparallel coiled-coil. Strikingly, the conserved lysine and arginine residues form a basic patch on one side of the structure, presenting a suitable docking surface for nucleic acids. Therefore, this repeated motif may represent a novel type of general RNA-binding domain appended to eukaryotic aminoacyl-tRNA synthetases to serve as a cis-acting tRNA-binding cofactor.

In the Nicotiana sylvestris CMSII mutant, a recombination-mediated change 5' to the first exon of the mitochondrial nad1 gene is associated with lack of the NADH:ubiquinone oxidoreductase (complex I) NAD1 subunit.

We previously reported that the Nicotiana sylvestris CMSII mutant mitochondrial DNA carried a large deletion. Several expressed sequences, most of which are duplicated, and the unique copy of the nad7 gene encoding the NAD7 subunit of the NADH:ubiquinone oxidoreductase complex (complex I) are found in the deletion. Here, we show that the orf87-nad3-nad1/A cotranscription unit transcribed from a unique promoter element in the wild-type, is disrupted in CMSII. Nad3, orf87 and the promoter element are part of the deleted sequence, whilst the nad1/A sequence is present and transcribed from a new promoter brought by the recombination event, as indicated by Northern and primer extension experiments. However, Western analyses of mitochondrial protein fractions and of complex I purified using anti-NAD9 affinity columns, revealed that NAD1 is lacking in CMSII mitochondria. Our results suggest that translation of nad1 transcripts rather than transcription itself could be altered in the mutant. Consequences of lack of this submit belonging the membrane arm of complex I and thought to contain the ubiquinone-binding site, are discussed.

Functional interaction of mammalian valyl-tRNA synthetase with elongation factor EF-1alpha in the complex with EF-1H.

In mammalian cells valyl-tRNA synthetase (ValRS) forms a high Mr complex with the four subunits of elongation factor EF-1H. The beta, gamma, and delta subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH2-terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1alpha could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1alpha and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNAVal synthesis catalyzed by the valyl-tRNA synthetase component of the ValRS.EF-1H complex. This effect is not observed in the presence of EF-1alpha and GDP or EF-Tu.GTP and requires association of valyl-tRNA synthetase within the ValRS.EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1alpha catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1alpha.GTP. Val-tRNAVal that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1alpha.GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells.

Macromolecular assemblage of aminoacyl-tRNA synthetases: identification of protein-protein interactions and characterization of a core protein.

In eukaryotes, from fly to human, nine aminoacyl-tRNA synthetases contribute a multienzyme complex of defined and conserved structural organization. This ubiquitous multiprotein assemblage comprises a unique bifunctional aminoacyl-tRNA synthetase, glutamyl-prolyl-tRNA synthetase, as well as the monospecific isoleucyl, leucyl, glutaminyl, methionyl, lysyl, arginyl, and aspartyl-tRNA synthetases. Three auxiliary proteins of apparent molecular masses of 18, 38 and 43 kDa are invariably associated with the nine tRNA synthetase components of the complex. As part of an inquiry into the molecular and functional organization of this macromolecular assembly, we isolated the cDNA encoding the p38 non-synthetase component and determined its function. The 320 amino acid residue encoded protein has been shown to have no homolog in yeast, bacteria and archaea, according to the examination of the complete genomic sequences available. The p38 protein is a moderately hydrophobic protein, displays a putative leucine-zipper motif, and shares a sequence pattern with protein domains that are involved in protein-protein interactions. We used the yeast two-hybrid system to register protein connections between components of the complex. We performed an exhaustive search of interactive proteins, involving 10 of the 11 components of the complex. Twenty-one protein pairs have been unambiguously identified, leading to a global view of the topological arrangement of the subunits of the multisynthetase complex. In particular, p38 was found to associate with itself to form a dimer, but also with p43, with the class I tRNA synthetases ArgRS and GlnRS, with the class II synthetases AspRS and LysRS, and with the bifunctional GluProRS. We generated a series of deletion mutants to localize the regions of p38 mediating the identified interactions. Mapping the interactive domains in p38 showed the specific association of p38 with its different protein partners. These findings suggest that p38, for which no homologous protein has been identified to date in organisms devoid of multisynthetase complexes, plays a pivotal role for the assembly of the subunits of the eukaryotic tRNA synthetase complex.

Modular evolution of the Glx-tRNA synthetase family--rooting of the evolutionary tree between the bacteria and archaea/eukarya branches.

The accuracy of protein biosynthesis generally rests on a family of 20 aminoacyl-tRNA synthetases, one for each amino acid. In bacteria, archaea and eukaryotic organelles, the formation of Gln-tRNA(Gln) is prevalently accomplished by a transamidation pathway, aminoacylation of tRNA(Gln) with Glu by glutamyl-tRNA synthetase (GluRS) followed by a tRNA-dependent transamidation of Glu from Glu-tRNA(Gln). A few bacterial species, such as Escherichia coli, possess a glutaminyl-tRNA synthetase (GlnRS), responsible for Gln-tRNA(Gln) formation. Phylogenetic analysis of the GluRS or GlnRS families (GlxRS) suggested that GlnRS has a eukaryotic origin and was horizontally transferred to a restricted set of bacteria. We have now isolated an additional GlnRS gene from the plant Lupinus luteus and analyzed in more details the modular architecture of the paralogous enzymes GluRS and GlnRS, starting from a large data set of 33 GlxRS sequences. Our analysis suggests that the ancestral GluRS-like enzyme was solely composed of the catalytic domain bearing the class-defining motifs of aminoacyl-tRNA synthetases, and that the anticodon-binding domain of GlxRSs was independently acquired in the bacteria and archaea branches of the universal tree of life, the eukarya sub-branch arising as a sister group of archaea. The transient capture of UAA and UAG codons could have favored the emergence of a GlnRS in early eukaryotes.

Switching the amino acid specificity of an aminoacyl-tRNA synthetase.

The accuracy of protein synthesis essentially rests on aminoacyl-tRNA synthetases that ensure the correct attachment of an amino acid to the cognate tRNA molecule. The selection of the amino acid substrate involves a recognition stage generally followed by a proofreading reaction. Therefore, to change the amino acid specificity of a synthetase in the aminoacylation reaction, it is necessary to alleviate the molecular barriers which contribute its editing function. In an attempt to accommodate a noncognate amino acid into the active site of a synthetase, we chose a pair of closely related enzymes. The current hypothesis designates glutaminyl-tRNA synthetase (GlnRS) as a late component of the protein synthesis machinery, emerging in the eukaryotic lineage by duplication of the gene for glutamyl-tRNA synthetase (GluRS). By introducing GluRS-specific features into the Rossmann dinucleotide-binding domain of human GlnRS, we constructed a mutant GlnRS which preferentially aminoacylates tRNA with glutamate instead of glutamine. Our data suggest that not only the transition state for aminoacyl-AMP formation but also the proofreading site of GlnRS are affected by that mutation.

Cloning and sequencing of cDNA encoding the rice methionyl-tRNA synthetase.

Three overlapping clones of cDNA, Mos43, Mos28 and Mos60, coding for methionyl-tRNA synthetase were obtained by screening the Oryza sativa lambda gt11 library. Their nucleotide sequence of 2850 bp was determined. The deduced amino-acid sequence of the isolated clones contains a HLGN and KFSKS motifs, which are conserved for this family of enzymes and have been proposed to be the signature sequences for class I aminoacyl-tRNA synthetases. A comparison of the rice MetRS primary structure with those deposited in EMBL/GenBank points to its high homology to yeast, human and Caenorhabditis elegans MetRSs. Interestingly, a great similarity of its C terminus to endothelial-monocyte-activating polypeptide II (EMAPII) and yeast protein G4p1 was observed.

Aspartyl-tRNA synthetase from rat: in vitro functional analysis of its assembly into the multisynthetase complex.

In mammalian cells, nine aminoacyl-tRNA synthetases, including aspartyl-tRNA synthetase, are associated within a multienzyme complex. Rat aspartyl-tRNA synthetase has a N-terminal polypeptide extension of about 40 amino acid residues which can be removed without impairing its catalytic activity. Earlier, in vivo studies showed that enzymes deprive of this N-terminal segment behave in vivo as free entities. We designed an experimental in vitro approach, based on the exchange of the complexed endogenous enzyme by free recombinant species, to assess the contribution of that domain in the association of aspartyl-tRNA synthetase to the complex. A phosphorylation site was introduced at the N-terminus of rat aspartyl-tRNA synthetase. The enzyme served as a reporter protein to evaluate the dissociation constants of native and N-terminal-truncated species towards the complex. Our data show that a moderate but significant drop in affinity is inferred by the removal of the N-terminal domain. The results suggest that this domain binds to another component of the complex, but might primarily serve a targeting function absolutely required in vivo for the assembly within the multienzyme structure.

The p43 component of the mammalian multi-synthetase complex is likely to be the precursor of the endothelial monocyte-activating polypeptide II cytokine.

p43 is one of the three auxiliary components invariably associated with nine aminoacyl-tRNA synthetases as a multienzyme complex ubiquitous to all eukaryotic cells from flies to humans. The cDNA encoding the hamster protein was isolated by using mixed oligonucleotides deduced from peptide sequences. The 359-amino acid protein is the hamster homologue of the recently reported murine and human EMAP II cytokine implicated in a variety of inflammatory disorders. The sequence of several proEMAP II proteins suggests that the p43 component of the complex is the precursor of the active mature cytokine after cleavage at a conserved Asp residue. The COOH-terminal moiety of p43 is also homologous to polypeptide domains found in bacterial methionyl- or phenylalanyl-tRNA synthetases and in the yeast Arc1p/G4p1 protein that associates with yeast methionyl-tRNA synthetase. Our results implicate the COOH-terminal moiety of p43 as a RNA binding domain. In the native state, as a component of the multisynthetase complex, p43 may be required for tRNA channeling and, after proteolytic processing occurring in tumor cells, would acquire inflammatory properties possibly related to apoptosis. The release of a truncated p43 from the complex could be involved in mediation of the signaling of tumor cells and stimulation of an acute inflammatory response.

Functional replacement of hamster lysyl-tRNA synthetase by the yeast enzyme requires cognate amino acid sequences for proper tRNA recognition.

We cloned the cDNA encoding a 597-aa hamster lysyl-tRNA synthetase. This enzyme is a close homologue of the 591-aa Saccharomyces cerevisiae enzyme, with the noticeable exception of their 60-aa N-terminal regions, which differ significantly. Several particular features of this polypeptide fragment from the hamster lysyl-tRNA synthetase suggest that it is implicated in the assembly of that enzyme within the multisynthetase complex. However, we show that this protein domain is dispensable in vivo to sustain growth of CHO cells. The cross-species complementation was investigated in the lysine system. The mammalian enzyme functionally replaces a null-allele of the yeast KRS1 gene. Conversely, the yeast enzyme cannot rescue Lys-101 cells, a CHO cell line with a temperature-sensitive lysyl-tRNA synthetase. The yeast and mammalian enzymes, overexpressed in yeast, were purified to homogeneity. The hamster lysyl-tRNA synthetase efficiently aminoacylates both mammalian and yeast tRNA(Lys), whereas the yeast enzyme aminoacylates mammalian tRNA(Lys) with a catalytic efficiency 20-fold lower, as compared to its cognate tRNA. The 152-aa C-terminus extremity of the hamster enzyme provides the yeast enzyme with the capacity to complement Lys-101 cells. This hybrid protein is fairly stable and aminoacylates both yeast and mammalian tRNA(Lys) with similar catalytic efficiencies. Because this C-terminal polypeptide fragment is likely to make contacts with the acceptor stem of tRNA(Lys), we conclude that it should carry the protein determinants conferring specific recognition of the cognate tRNA acceptor stem and therefore contributes an essential role in the operational RNA code for amino acids.

Genomic organization of the rat aspartyl-tRNA synthetase gene family: a single active gene and several retropseudogenes.

The genomic organization of the gene encoding rat aspartyl-tRNA synthetase (AspRS), a class II aminoacyl-tRNA synthetase (aaRS), was determined. A single active gene and several pseudogenes were isolated from a rat genomic DNA library and characterized. The active DRS1 gene encoding the rat AspRS spans approximately 60 kb and is divided into 16 exons. Exons 8-16, encoding the nt-binding domain of the synthetase, are clustered in the 3'-region of the gene, whereas exons 3, 4, and 5, encoding the anticodon-binding domain are separated by large introns (up to 15 kb) containing LINE sequences. One of the pseudogenes, psi DRS1, has a nt sequence 93% identical to that of the complete cDNA sequence of rat AspRS but several stop codons interrupt the coding sequence, thus identifying psi DRS1 to an inactive processed pseudogene. Two repetitive elements from the LINE family are inserted into psi DRS1. Calculation of nt substitution rates suggests that psi DRS1 sequences arose approximately 27 Myr ago. The other pseudogene, psi DRS2, should be more ancient. Taken together, these results clearly demonstrate that the AspRS gene family is composed of only one active gene. The availability of the gene structure of AspRS could help to clarify molecular evolution of class II aaRS.

Expression of rat aspartyl-tRNA synthetase in Saccharomyces cerevisiae. Role of the NH2-terminal polypeptide extension on enzyme activity and stability.

Cytoplasmic aspartyl-tRNA synthetase from mammals is one of the components of a multienzyme complex comprising nine synthetase activities. The presence of an amino-terminal extension composed of about 40 residues is a characteristic of the eukaryotic enzyme. We report here the expression in the yeast Saccharomyces cerevisiae of a native form of rat aspartyl-tRNA synthetase and of two truncated derivatives lacking 20 or 36 amino acid residues from their amino-terminal polypeptide extension. The three recombinant enzyme species were purified to homogeneity. They behave as alpha2 dimers and display catalytic parameters in the tRNA aminoacylation reaction identical to those determined for the native, complex-associated form of aspartyl-tRNA synthetase isolated from rat liver. Because the dimer dissociation constant of rat AspRS is much higher than that of its bacterial and yeast counterparts, we could establish a direct correlation between dissociation of the dimer and inactivation of the enzyme. Our results clearly show that the monomer is devoid of amino acid activation and tRNA aminoacylation activities, indicating that dimerization is essential to confer an active conformation on the catalytic site. The two NH2-terminal truncated derivatives were fully active, but proved to be more unstable than the recombinant native enzyme, suggesting that the polypeptide extension fulfills structural rather than catalytic requirements.