A component of the multisynthetase complex is a multifunctional aminoacyl-tRNA synthetase.

Abstract

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.

Evolution of The Aminoacyl‐tRNa Synthetase Family and The Organization of the Drosophila Glutamyl‐Prolyl‐tRNA Synthetase Gene — Intron/Exon Structure of the Gene, Control of Expression of the Two mRNAs, Selective Advantage of the Multienzyme Complex

“…Genomic clones were isolated by standard protocols (Sambrook et al, 1989) from an EMBL3 library made from Drosophilu melanoguster Canton S adult flies (Clontech Laboratories). Three fragments covering the total length (5.3 kb) of the longest cDNA clone A 2 7 (Cerini et al, 1991) and corresponding respectively to the N-terminal, the repeated motifs and the C-terminal coding sequence were used as hybridization probes. They allowed the isolation of 19 kb of genomic DNA contained in two overlapping phages.…”

“…cDNAs encoding the PGI protein (Cerini et al, 1991), containing essentially the glutamyl domain and the repeated motifs, and that encoding only the repeated motifs domain were subcloned in the pGEX-2T vector (Pharmacia). In both cases, the proteins were prepared according to the manufacturer's instructions and used as immunogens to elicit the production of specific antisera in mice as described by Harlow and Lane (1988).…”

“…The two synthetase domains are separated by a spacer region constituted of units repeated six times. Each unit consists of a core of 46 highly conserved amino acids named the repeated motifs (RM) and of 26-33 more variable residues (Cerini et al, 1991). The glutamyl domain can be classified as a class I aminoacyl-tRNA synthetase while the three motifs of class I1 enzymes are found in the C-terminal domain.…”

“…As described by Cerini et al (1991), two mRNAs are transcribed from the Drosophilu glutamyl-prolyl-tRNA synthetase gene. While the longest one (6.1 kb) coding for the full-length protein is present at all stages of development, the shorter transcript (3.4 kb) encompassing the 3'-end of the cDNA is especially abundant in 5-10-h embryos and remains at the same level until the first larval stage.…”

“…A single copy of the repeated motif exists in the N-terminal position of Trp-tRNA and His-tRNA synthetases which are proteins that never associate in the complexes (Tsui and Siminovitch, 1987;Frolova et al, 1991;Timchenko and Caskey, 1994). In fact, amino acid substitutions in the consensus sequence of these single-copy motifs suggest that they cannot play the same role as in the complex (Cerini et al, 1991). These internal repeated units present as highly organized structures seem to have appeared concomitantly to gene fusion and are encountered only in proteins that are associated within highmolecular-mass complexes.…”