Publications

• Non-discriminating and discriminating aspartyl-tRNA synthetases differ in the anticodon-binding domain.

  Charron C., Roy H., Blaise M., Giege R., Kern D.

  In most organisms, tRNA aminoacylation is ensured by 20 aminoacyl-tRNA synthetases (aaRSs). In eubacteria, however, synthetases can be duplicated as in Thermus thermophilus, which contains two distinct AspRSs. While AspRS-1 is specific, AspRS-2 is non-discriminating and aspartylates tRNA(Asp) and ... >> More

  In most organisms, tRNA aminoacylation is ensured by 20 aminoacyl-tRNA synthetases (aaRSs). In eubacteria, however, synthetases can be duplicated as in Thermus thermophilus, which contains two distinct AspRSs. While AspRS-1 is specific, AspRS-2 is non-discriminating and aspartylates tRNA(Asp) and tRNA(Asn). The structure at 2.3 A resolution of AspRS-2, the first of a non-discriminating synthetase, was solved. It differs from that of AspRS-1 but has resemblance to that of discriminating and archaeal AspRS from Pyrococcus kodakaraensis. The protein presents non-conventional features in its OB-fold anticodon-binding domain, namely the absence of a helix inserted between two beta-strands of this fold and a peculiar L1 loop differing from the large loops known to interact with tRNA(Asp) identity determinant C36 in conventional AspRSs. In AspRS-2, this loop is small and structurally homologous to that in AsnRSs, including conservation of a proline. In discriminating Pyrococcus AspRS, the L1 loop, although small, lacks this proline and is not superimposable with that of AspRS-2 or AsnRS. Its particular status is demonstrated by a loop-exchange experiment that renders the Pyrococcus AspRS non-discriminating. << Less

  EMBO J. 22:1632-1643(2003) [PubMed] [EuropePMC]

• Toward the full set of human mitochondrial aminoacyl-tRNA synthetases: characterization of AspRS and TyrRS.

  Bonnefond L., Fender A., Rudinger-Thirion J., Giege R., Florentz C., Sissler M.

  The human mitochondrion possesses a translational machinery devoted to the synthesis of 13 proteins. While the required tRNAs and rRNAs are produced by transcription of the mitochondrial genome, all other factors needed for protein synthesis are synthesized in the cytosol and imported. This is the ... >> More

  The human mitochondrion possesses a translational machinery devoted to the synthesis of 13 proteins. While the required tRNAs and rRNAs are produced by transcription of the mitochondrial genome, all other factors needed for protein synthesis are synthesized in the cytosol and imported. This is the case for aminoacyl-tRNA synthetases, the enzymes which esterify their cognate tRNA with the specific amino acid. The genes for the full set of cytosolic aaRSs are well defined, but only nine genes for mitochondrial synthetases are known. Here we describe the genes for human mitochondrial aspartyl- and tyrosyl-tRNA synthetases and the initial characterization of the enzymes. Both belong to the expected class of synthetases, have a dimeric organization, and aminoacylate Escherichia coli tRNAs as well as in vitro transcribed human mitochondrial tRNAs. Genes for the remaining missing synthetases were also found with the exception of glutaminyl-tRNA synthetase. Their sequence analysis confirms and further extends the view that, except for lysyl- and glycyl-tRNA synthetases, human mitochondrial and cytosolic enzymes are coded by two different sets of genes. << Less

  Biochemistry 44:4805-4816(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Expanding tRNA recognition of a tRNA synthetase by a single amino acid change.

  Feng L., Tumbula-Hansen D., Toogood H., Soll D.

  Aspartyl-tRNA synthetase (AspRS) occurs in two types: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), whereas the nondiscriminating enzyme (ND-AspRS) also synthesizes Asp-tRNA(Asn), which is a required intermediate for protein synthesis in many organisms. We attempted to expand the t ... >> More

  Aspartyl-tRNA synthetase (AspRS) occurs in two types: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), whereas the nondiscriminating enzyme (ND-AspRS) also synthesizes Asp-tRNA(Asn), which is a required intermediate for protein synthesis in many organisms. We attempted to expand the tRNA recognition of the discriminating Thermococcus kodakaraensis AspRS to that of a ND-AspRS by in vitro mutagenesis. An alignment of 26 archaeal AspRS proteins revealed two positions (26 and 85 in the T. kodakaraensis sequence) whose amino acid identity changes according to the enzymes' tRNA specificity. In their anticodon-binding domain, D-AspRS proteins contain W26 (or Q26) and K85, compared with H26 and P85 in the ND-AspRSs. T. kodakaraensis AspRS gained the ability to form Asp-tRNA(Asn) in vitro when the W26H or K85P changes were introduced independently or in combination. In the aminoacylation of tRNA(Asn) or tRNA(Asp) transcripts, the mutant enzymes displayed at least a 100-to 500-fold change in tRNA specificity, as judged by the ratio of the k(cat)K(m) values of Asp-tRNA(Asp) vs. Asp-tRNA(Asn) formation. That T. kodakaraensis mutant AspRSs mischarge tRNA(Asn) was also manifested in the higher level (1.7%) of aspartylation of unfractionated Pyrococcus tRNA compared with that achieved by the wild-type enzyme (0.9%). Northern blot analysis of the Asp-tRNA separated by acidurea gel electrophoresis confirmed the in vitro synthesis of Asp-tRNA(Asn). A structure-based model points to a direct interaction of K85 in T. kodakaraensis AspRS with the anticodon nucleotide C36 of tRNA(Asp). Thus, a switch between D-AspRS and ND-AspRS enzymes could have evolved with only limited amino acid changes. << Less

  Proc. Natl. Acad. Sci. U.S.A. 100:5676-5681(2003) [PubMed] [EuropePMC]

• Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture.

  Neuenfeldt A., Lorber B., Ennifar E., Gaudry A., Sauter C., Sissler M., Florentz C.

  In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This r ... >> More

  In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This raises the question of molecular adaptation of these proteins to their peculiar partner RNAs. The crystal structure of the homodimeric bacterial-type human mitochondrial aspartyl-tRNA synthetase (DRS) confirmed a 3D architecture close to that of Escherichia coli DRS. However, the mitochondrial enzyme distinguishes by an enlarged catalytic groove, a more electropositive surface potential and an alternate interaction network at the subunits interface. It also presented a thermal stability reduced by as much as 12°C. Isothermal titration calorimetry analyses revealed that the affinity of the mitochondrial enzyme for cognate and non-cognate tRNAs is one order of magnitude higher, but with different enthalpy and entropy contributions. They further indicated that both enzymes bind an adenylate analog by a cooperative allosteric mechanism with different thermodynamic contributions. The larger flexibility of the mitochondrial synthetase with respect to the bacterial enzyme, in combination with a preserved architecture, may represent an evolutionary process, allowing nuclear-encoded proteins to cooperate with degenerated organelle RNAs. << Less

  Nucleic Acids Res. 41:2698-2708(2013) [PubMed] [EuropePMC]

• Existence of two distinct aspartyl-tRNA synthetases in Thermus thermophilus. Structural and biochemical properties of the two enzymes.

  Becker H.D., Reinbolt J., Kreutzer R., Giege R., Kern D.

  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 ... >> More

  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. << Less

  Biochemistry 36:8785-8797(1997) [PubMed] [EuropePMC]

• Thermus thermophilus contains an eubacterial and an archaebacterial aspartyl-tRNA synthetase.

  Becker H.D., Roy H., Moulinier L., Mazauric M.H., Keith G., Kern D.

  Thermus thermophilus possesses two aspartyl-tRNA synthetases (AspRSs), AspRS1 and AspRS2, encoded by distinct genes. Alignment of the protein sequences with AspRSs of other origins reveals that AspRS1 possesses the structural features of eubacterial AspRSs, whereas AspRS2 is structurally related t ... >> More

  Thermus thermophilus possesses two aspartyl-tRNA synthetases (AspRSs), AspRS1 and AspRS2, encoded by distinct genes. Alignment of the protein sequences with AspRSs of other origins reveals that AspRS1 possesses the structural features of eubacterial AspRSs, whereas AspRS2 is structurally related to the archaebacterial AspRSs. The structural dissimilarity between the two thermophilic AspRSs is correlated with functional divergences. AspRS1 aspartylates tRNA(Asp) whereas AspRS2 aspartylates tRNA(Asp), and tRNA(Asn) with similar efficiencies. Since Asp bound on tRNA(Asn) is converted into Asn by a tRNA-dependent aspartate amidotransferase, AspRS2 is involved in Asn-tRNA(Asn) formation. These properties relate functionally AspRS2 to archaebacterial AspRSs. The structural basis of the dual specificity of T. thermophilus tRNA(Asn) was investigated by comparing its sequence with those of tRNA(Asp) and tRNA(Asn) of strict specificity. It is shown that the thermophilic tRNA(Asn) contains the elements defining asparagine identity in Escherichia coli, part of which being also the major elements of aspartate identity, whereas minor elements of this identity are missing. The structural context that permits expression of aspartate and asparagine identities by tRNA(Asn) and how AspRS2 accommodates tRNA(Asp) and tRNA(Asn) will be discussed. This work establishes a distinct structure-function relationship of eubacterial and archaebacterial AspRSs. The structural and functional properties of the two thermophilic AspRSs will be discussed in the context of the modern and primitive pathways of tRNA aspartylation and asparaginylation and related to the phylogenetic connexion of T. thermophilus to eubacteria and archaebacteria. << Less

  Biochemistry 39:3216-3230(2000) [PubMed] [EuropePMC]

• Aminoacyl-tRNA synthesis.

  Ibba M., Soll D.

  Aminoacyl-tRNAs are substrates for translation and are pivotal in determining how the genetic code is interpreted as amino acids. The function of aminoacyl-tRNA synthesis is to precisely match amino acids with tRNAs containing the corresponding anticodon. This is primarily achieved by the direct a ... >> More

  Aminoacyl-tRNAs are substrates for translation and are pivotal in determining how the genetic code is interpreted as amino acids. The function of aminoacyl-tRNA synthesis is to precisely match amino acids with tRNAs containing the corresponding anticodon. This is primarily achieved by the direct attachment of an amino acid to the corresponding tRNA by an aminoacyl-tRNA synthetase, although intrinsic proofreading and extrinsic editing are also essential in several cases. Recent studies of aminoacyl-tRNA synthesis, mainly prompted by the advent of whole genome sequencing and the availability of a vast body of structural data, have led to an expanded and more detailed picture of how aminoacyl-tRNAs are synthesized. This article reviews current knowledge of the biochemical, structural, and evolutionary facets of aminoacyl-tRNA synthesis. << Less

  Annu Rev Biochem 69:617-650(2000) [PubMed] [EuropePMC]

  This publication is cited by 26 other entries.