Abstract
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.
Highlights
Aminoacyl-tRNA synthetases are essential enzymes that catalyze a key step during the process of protein biosynthesis: activation of a specific amino acid and the charging of the cognate tRNA (1)
The dissociation of rat Aspartyl-tRNA synthetase (AspRS) and the concomitant loss of both amino acid activation and tRNA aminoacylation activities establish for the first time that dimerization of AspRS, a representative of class IIb synthetases, is absolutely required for its catalytic activity
Our results are in agreement with those previously reported for a class IIa synthetase, Pro-tRNA synthetase from E. coli, where dimerization has been shown to be required for activity (44)
Summary
Strains and Plasmids—M13mp, the yeast shuttle vectors pYeDP1/ 8-10 and pYeDP60 Construction of the Expression Vector pYeDP60/DRS for Overproduction of Rat AspRS in S. cerevisiae—An EcoRI-EcoRI cDNA fragment containing the complete coding sequence of the rat AspRS cDNA (19) was cloned into the EcoRI site of pYeDP1/8-10 (24) to give pYeDP1/ DRS This plasmid has the yeast 2-m origin of replication and the URA3-selectable marker but displays a poor mitotic stability. Construction of pYeDP60/DRS-⌬N20 —We produced a DNA fragment by amplification of M13mp9/DRS1 between the oligonucleotides 5Ј-GGGATCCTTCTCGAGTATGATACAATCACAAG-3Ј (F01) and DRS666IC (22) This corresponds to DRS1 cDNA over positions 101– 666 and allows the insertion of a XhoI site in the coding sequence of rat AspRS. Fractions containing AspRS activity (eluted at 110 mM) were pooled, diluted 5-fold with 10% glycerol and 1 mM DTE, and applied to a 1.1 ϫ 8.5-cm column of tRNA-Sepharose (29) equilibrated in 25 mM potassium phosphate (pH 7.5), 10% glycerol, 1 mM DTE. Since inclusion of a second virial coefficient gave no improvement of the fit, an ideal component system was considered
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