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In higher eukaryotes, nine aminoacyl-tRNA synthetases are associated within a multienzyme complex which is composed of 11 polypeptides with molecular masses ranging from 18 to 150 kDa. We have cloned and sequenced a cDNA from Drosophila encoding the largest polypeptide of this complex. We demonstrate here that the corresponding protein is a multifunctional aminoacyltRNA synthetase. It is composed of three major domains, two of them specifying distinct synthetase activities. The amino and carboxy-terminal domains were expressed separately in Escherichia coli, and were found to catalyse the aminoacylation of glutamic acid and proline tRNA species, respectively. The central domain is made of six 46 amino acid repeats. In prokaryotes, these two aminoacyl-tRNA synthetases are encoded by distinct genes. The emergence of a multifunctional synthetase by a gene fusion event seems to be a specific, but general attribute of all higher eukaryotic cells. This type of structural organization, in relation to the occurrence of multisynthetase complexes, could be a mechanism to integrate several catalytic domains within the same particle. The involvement of the internal repeats in mediating complex assembly is discussed.

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 in¯ammatory 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 A Ê 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 tRNAbinding 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.

In mammalian cells valyl-tRNA synthetase (ValRS) forms a high M r complex with the four subunits of elongation factor EF-1H. The ␤, ␥, and ␦ subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH 2 -terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1␣ could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1␣ and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNA Val synthesis catalyzed by the valyl-tRNA synthetase component of the ValRS⅐EF-1H complex. This effect is not observed in the presence of EF-1␣ and GDP or EF-Tu⅐GTP and requires association of valyl-tRNA synthetase within the ValRS⅐EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1␣ catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1␣⅐GTP⅐Val-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-1␣⅐GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells.Aminoacyl-tRNA is the donor of amino acid in ribosomal protein synthesis. The tRNA molecule is aminoacylated with the corresponding amino acid by an aminoacyl-tRNA synthetase, the aminoacyl-tRNA is converted to a ternary complex with elongation factor 1␣, to give the immediate precursor of amino acid for protein synthesis: EF-1␣⅐GTP⅐aminoacyl-tRNA. Several lines of evidence have suggested that in mammalian cells the translational apparatus is highly organized. In particular, association of the protein vectors of tRNA, aminoacyltRNA synthetases and elongation factors, with the cytoskeletal framework has been reported (1-3) and colocalization of these components described (4). The isolation and characterization of supramolecular assemblies of aminoacyl-tRNA synthetases and elongation factors (5-7) have provided structural evidence for the subcellular organization of the protein synthesis machinery. The existence of a channeled tRNA cycle during mammalian protein synthesis provided functional evidence for cellular compartmentalization of translation (8 -10). According to the proposed channeling scheme, aminoacyl-tRNAs are vectorially transferred from the aminoacyl-tRNA synthetases to the ribosomes as ternary complexes EF-1␣⅐GTP⅐aminoacyl-tRNA (8, 10). Moreover, the GDP form of EF-1␣ could be involved in the capture of deacylated tRNA at the exit site of the ribosome and its delivery to the synthetase (11).Channeling, or direct transfer of metabolites from one enzyme to another in a metabolic pathway, is believed to increase significantly the efficiency of the overall reaction (12). For sequential metabolic enzymes, the stimulation of activity of the first enzyme induced by a protein-protein interactio...

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In Saccharomyces cerevisiae, the expression of several genes implicated in methionine biosynthesis is co-regulated by a specific negative control. To elucidate the molecular basis of this regulation, we have cloned two of these genes, MET3 and MET25. The sequence of MET25 has already been determined (Kerjan et al. 1986). Here, we report the nucleotide sequence of the MET3 gene along with its 5' and 3' flanking regions. Plasmids bearing different deletions upstream of the transcribed region of MET3 were constructed. They were introduced into yeast cells and tested for their ability to complement met3 mutations and to respond to regulation by exogenous methionine. The regulatory region was located within a 100 bp region. The sequence of this regulatory region was compared with that of MET25. A short common sequence which occurs 250-280 bp upstream of the translation initiation codon of the gene was found. This sequence is a good candidate for the cis-acting regulatory element.

The respective contributions of electrostatic interaction and specific sequence recognition in the binding of microtubule-associated proteins (MAPs) to microtubules have been studied, using as models yeast valyl- and lysyl-tRNA synthetases (VRS, KRS) that carry an exposed basic N-terminal domain, and a synthetic peptide reproducing the sequence 218-235 on tau protein, known to be part of the microtubule-binding site of MAPs. VRS and KRS bind to microtubules with a KD in the 10(-6) M range, and tau 218-235 binds with a KD in the 10(-4) M range. Binding of KRS and tau 218-235 is accompanied by stabilization and bundling of microtubules, without the intervention of an extraneous bundling protein. tau 218-235 binds to microtubules with a stoichiometry of 2 mol/mol of assembled tubulin dimer in agreement with the proposed binding sequences alpha[430-441] and beta[422-434]. Binding stoichiometries of 2/alpha beta S tubulin and 1/alpha S beta S tubulin were observed following partial or complete removal of the tubulin C-terminal regions by subtilisin, which localizes the site of subtilisin cleavage upstream residue alpha-441 and downstream residue beta-434. Quantitative measurements show that binding of MAPs, KRS, VRS, and tau 218-235 is weakened but not abolished following subtilisin digestion of the C-terminus of tubulin, indicating that the binding site of MAPs is not restricted to the extreme C-terminus of tubulin.