Two aspartyl-tRNA synthetases (AspRSs) were isolated from Thermus thermophilus HB8. Both are alpha2 dimers but differ in the length of their polypeptide chains (AspRS1, 68 kDa; and AspRS2, 51 kDa). Both chains start with Met and are deprived of common sequences to a significant extent. This rules out the possibility that AspRS2 is derived from AspRS1 by proteolysis, in agreement with specific recognition of each AspRS by the homologous antibodies. DNA probes derived from N-terminal amino acid sequences hybridize specifically to different genomic DNA fragments, revealing that the two AspRSs are encoded by distinct genes. Both enzymes are present in various strains from T. thermophilus and along the growth cycle of the bacteria, suggesting that they are constitutive. Kinetic investigations show that the two enzymes are specific for aspartic acid activation and tRNAAsp charging. tRNA aspartylation by the thermostable AspRSs is governed by thermodynamic parameters which values are similar to those measured for mesophilic aspartylation systems. Both thermophilic AspRSs are deprived of species specificity for tRNA aspartylation and exhibit N-terminal sequence signatures found in other AspRSs, suggesting that they are evolutionarily related to AspRSs from mesophilic prokaryotes and eukaryotes. Comparison of the efficiency of tRNA aspartylation by each enzyme under conditions approaching the physiological ones suggests that in vivo tRNAAsp charging is essentially ensured by AspRS1, although AspRS2 is the major species. The physiological significance of the two different AspRSs in T. thermophilus is discussed.

His85 in Thermus thermophilus elongation factor Tu (EF-Tu) was replaced by glutamine, leucine and glycine residues, leading to [H85Q]EF-Tu, [H85L] EF-Tu and [H85G]EF-Tu, respectively. Asp81 was replaced by alanine leading to [D81A]EF-Tu, and replacement of Arg300 provided [R300I]EF-Tu. Glycine in position 85 of domain I induces a protease-sensitive site in domain II and causes complete protein degradation in vivo. A similar effect was observed when Asp81 was replaced by alanine or Arg300 by isoleucine. Degradation is probably due to disturbed interactions between the domains of EF-Tu.GTP, inducing a protease-sensitive cleavage site in domain II. [H85Q]EF-Tu, which can be effectively overproduced in Escherichia coli, is slower in poly(U)-dependent poly(Phe) synthesis, has lower affinity to aminoacyl-tRNA but shows only a slightly reduced rate of intrinsic GTP hydrolysis compared to the native protein. The GTPase of this protein variant is not efficiently stimulated by aminoacyl-tRNA and ribosomes. The slow GTPase of [H85Q]EF-Tu increases the fidelity of translation as measured by leucine incorporation into poly(Phe) in in vitro poly(U)-dependent ribosomal translation. Replacement of His85 in T. thermophilus EF-Tu by leucine completely deactivates the GTPase activity but does not substantially influence the aminoacyl-tRNA binding. [H85L]EF-Tu is inactive in poly(U)-dependent poly(Phe)-synthesis. The rate of nucleotide dissociation is highest for [H85L]EF-Tu, followed by [H85Q]EF-Tu and native T. thermophilus EF-Tu. Mutation of His85, a residue which is not directly involved in the nucleotide binding, thus influences the interaction of EF-Tu domains, nucleotide binding and the efficiency and rate of GTPase activity.

The tRNA glycylation system is amongst the most complex aminoacylation systems since neither the oligomeric structure of the enzymes nor the discriminator base in tRNAs are conserved in the phylae. To understand better this structural diversity and its functional consequences, the prokaryotic glycylation system from Thermus thermophilus, an extreme thermophile, was investigated and its structural and functional inter-relations with those of other origins analyzed. Alignments of the protein sequence of the dimeric thermophilic glycyl-tRNA synthetase (Gly-tRNA synthetase) derived from its gene with sequences of other dimeric Gly-tRNA synthetases revealed an atypical character of motif 1 in all these class 2 synthetases. Interestingly, the sequence of the prokaryotic thermophilic enzyme resembles eukaryotic and archaebacterial Gly-tRNA synthetases, which are all dimeric, and diverges drastically from the tetrameric enzymes from other prokaryotes.Cross aminoacylations with tRNAs and synthetases of different origins provided information about functional interrelations between the glycylation systems. Efficient glycylations involving partners from T. thermophilus and Escherichia coli showed conservation of the recognition process in prokaryotes despite strong structural variations of the synthetases. However, Gly-tRNA synthetase from T. thermophilus acylates eukaryotic tRNA Gly while the charging ability of the E. coli enzyme is restricted to prokaryotic tRNA Gly . A similar behaviour is found in eukaryotic systems where the restricted species specificity for tRNA glycylation of mammalian Gly-tRNA synthetase contrasts with the relaxed specificity of the yeast enzyme. The consensus sequence of the tRNAs charged by the various Gly-tRNA synthetases reveals conservation of only G1ϪC72 in the acceptor arm, C35 and C36 in the anticodon, and the (G10Ϫ Y25)-G45 triplet involved in tRNA folding. Conservation of these nucleotides indicates their key role in glycylation and suggests that they were part of the ancestral glycine identity set.These features are discussed in the context of the phylogenic connections between prokaryotes, eukaryotes, and archaebacteria, and of the particular place of T. thermophilus in this phylogeny.