AN EXAMPLE OF NONCONSERVATION OF OLIGOMERIC STRUCTURE IN PROKARYOTIC AMINOACYL-TRANSFER-RNA SYNTHETASES - BIOCHEMICAL AND STRUCTURAL-PROPERTIES OF GLYCYL-TRANSFER-RNA SYNTHETASE FRONT THERMUS-THERMOPHILUS

Glycyl-tRNA synthetase (Gly-tRNA synthetase) from Thermus thermophilus was purified to homogeneity and with high yield using a five-step pur ification procedure in amounts sufficient to solve its crystallographi c structure [Logan, D. T., Mazauric, M.-H., Kern, D. & Moras, D. (1995 ) EMBO J. 14, 4156-4167]. Molecular-mass determinations of the native and denatured protein indicate an oligomeric structure of the alpha(2) type consistent with that found for eukaryotic Gly-tRNA synthetases ( yeast and Bombyx mori), but different from that of Gly-tRNA synthetase s from mesophilic prokaryotes (Escherichia coli and Bacillus brevis) w hich are alpha(2) beta(2) tetramers. N-terminal sequencing of the poly peptide chain reveals significant identity, reaching 50% with those of the eukaryotic enzymes (B. mori, Homo sapiens, yeast and Caenorhabdit is elegans) but no significant identity was found with both alpha and beta chains of the prokaryotic enzymes (E. coli, Haemophilus influenza e and Coxiella burnetti) albeit the enzyme is deprived of the N-termin al extension characterizing eukaryotic synthetases. Thus, the thermoph ilic Gly-tRNA synthetase combines strong structural homologies of euka ryotic Gly-tRNA synthetases with a feature of prokaryotic synthetases. Heat-stability measurements show that this synthetase keeps its ATP-P P, exchange and aminoacylation activities up to 70 degrees C. Glycylad enylate strongly protects the enzyme against thermal inactivation at h igher temperatures. Unexpectedly, tRNA(Gly) does not induce protection . Cross-aminoacylations reveal that the thermophilic Gly-tRNA syntheta se charges heterologous E. coli tRNA(Gly(GCC)) and tRNA(Gly(GCC)) and yeast tRNA(Gly(CCC)) as efficiently as T. thermophilus tRNA(Gly). All these aminoacylation reactions are characterized by similar activation energies as deduced from Arrhenius plots. Therefore, contrary to the E. coli and H. sapiens Gly-tRNA synthetases, the prokaryotic thermophi lic enzyme does not possess a strict species specificity. The results are discussed in the context of the three-dimensional structure of the synthetase and in the view of the particular evolution of the glyciny lation systems.