Crystallization of threonyl‐tRNA synthetase from <i>Thermus thermophilus</i> and preliminary crystallographic data

Cura, Vincent; Kern, Daniel; Mitschler, A.; Moras, Dino

doi:10.1016/0014-5793(95)01089-w

Cited by 6 publications

(1 citation statement)

References 24 publications

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“…The overexpressed enzyme exhibits a specific activity of tRNA aminoacylation at 70 °C of 340 nmol·min −1 ·mg −1 ( k cat = 0.43·s −1 ), identical to that of the enzyme isolated from T. thermophilus (354 nmol·min −1 ·mg −1 ) [45]. The overexpressed protein crystallizes from solutions containing ammonium sulfate and glycerol [66]. Analysis by electrospray mass spectrometry revealed two enzyme populations of M r 75 550.87 ± 10.76 and 75 442.30 ± 17.74.…”

Section: Resultsmentioning

confidence: 99%

Sequence analysis and modular organization of threonyl‐tRNA synthetase from Thermus thermophilus and its interrelation with threonyl‐tRNA synthetases of other origins

Cura

Moras

Kern

2000

European Journal of Biochemistry

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The gene encoding threonyl-tRNA synthetase (Thr-tRNA synthetase) from the extreme thermophilic eubacterium Thermus thermophilus HB8 has been cloned and sequenced. The ORF encodes a polypeptide chain of 659 amino acids (M r 75 550) that shares strong similarities with other Thr-tRNA synthetases. Comparative analysis with the three-dimensional structure of other subclass IIa synthetases shows it to be organized into four structural modules: two N-terminal modules specific to Thr-tRNA synthetases, a catalytic core and a C-terminal anticodonbinding module. Comparison with the three-dimensional structure of Escherichia coli Thr-tRNA synthetase in complex with tRNA Thr enabled identification of the residues involved in substrate binding and catalytic activity. Analysis by atomic absorption spectrometry of the enzyme overexpressed in E. coli revealed the presence in each monomer of one tightly bound zinc atom, which is essential for activity.Despite strong similarites in modular organization, Thr-tRNA synthetases diverge from other subclass IIa synthetases on the basis of their N-terminal extensions. The eubacterial and eukaryotic enzymes possess a large extension folded into two structural domains, N1 and N2, that are not significantly similar to the shorter extension of the archaebacterial enzymes. Investigation of a truncated Thr-tRNA synthetase demonstrated that domain N1 is not essential for tRNA charging. Thr-tRNA synthetase from T. thermophilus is of the eubacterial type, in contrast to other synthetases from this organism, which exhibit archaebacterial characteristics. Alignments show conservation of part of domain N2 in the C-terminal moiety of Ala-tRNA synthetases. Analysis of the nucleotide sequence upstream from the ORF showed the absence of both any anticodon-like stem-loop structure and a loop containing sequences complementary to the anticodon and the CCA end of tRNA Thr . This means that the expression of Thr-tRNA synthetase in T. thermophilus is not regulated by the translational and trancriptional mechanisms described for E. coli thrS and Bacillus subtilis thrS and thrZ. Here we discuss our results in the context of evolution of the threonylation systems and of the position of T. thermophilus in the phylogenic tree.Keywords: threonyl-tRNA synthetase, tRNA aminoacylation, evolution of aminoacylation systems, Thermus thermophilus, extremophiles.Fidelity of translation depends upon accurate aminoacylation of tRNAs by aminoacyl-tRNA synthetases. Most organisms possess 20 synthetases [1±4], each aminoacylating its cognate tRNA with the homologous amino acid via a two-step process in which the amino acid is activated before being transferred on to tRNA. This reaction does not occur with absolute specificity, however, and to reduce error levels synthetases developed kinetic artifices ensuring selection of the cognate substrates and destruction of wrong end-products [5±8].The complexity of the aminoacylation system at the functional level is reinforced by the structural diversity of aminoacyl-tRNA synthetase...

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Section: Resultsmentioning

confidence: 99%

Sequence analysis and modular organization of threonyl‐tRNA synthetase from Thermus thermophilus and its interrelation with threonyl‐tRNA synthetases of other origins

Cura

Moras

Kern

2000

European Journal of Biochemistry

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A view into the origin of life: aminoacyl-tRNA synthetases

Pouplana¹,

Schimmel²

2000

CMLS, Cell. Mol. Life Sci.

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The genetic code converts nucleic acid sequences into protein sequences through the action of aminoacyltransfer RNA (tRNA) synthetases. These enzymes are responsible for the specific attachment of each amino acid to its cognate tRNAs. The aminoacylated tRNAs are then transported to the ribosomal complex by elongation factors, where the amino acid is incorporated into the translated polypeptide chain. The RNA world hypothesis states that modern aminoacyl-tRNA synthetases replaced aminoacylating ribozymes. From this perspective, the establishment of modern aminoacyltRNA synthetases would span the transition from the RNA world to the world of DNA and proteins. Thus, the study of the evolution of aminoacyl-tRNA synthetases could offer insights into that process. Here we review our current understanding of the origin and evolution of aminoacyl-tRNA synthetases, and discuss the implications of these studies on the origin of life. The RNA backgroundThe theory of an RNA world postulates that life evolved through a stage where all functions currently performed by proteins and DNA depended chiefly on RNA [1]. From physical and chemical considerations, this RNA-based metabolism has been proposed to be preceded by a 'chemical world', perhaps based on surface-based catalytic processes [2 -6]. The products of these reactions laid the foundation for the eventual appearance of RNA-based protogenomes and membranes, essential requirements for the establishment of populations subjected to natural selection. The categorical demonstration of the existence of an RNA world is impossible without the discovery of an extant form of RNA-based organisms. The probability of such discovery being made is small because one would expect such organisms to have been eliminated by competing species bearing protein-based physiologies. Thus, the theory of an RNA world springs out of theoretical considerations based on three observations: the central role of RNA and ribonucleoproteins in decoding genetic information [9]; the use of catalytic RNA molecules in limited aspects of modern cell metabolism [10, 11]; the potential of in vitro selected RNA molecules to catalyze important biochemical reactions [12][13][14]. These considerations all point to RNA as a plausible transition molecule, a jack-of-all-trades that was capable of undertaking the necessary functions required for establishment of a primitive cell [7,15]. Eventually, the superior stability of DNA, and the greater catalytic plasticity and capacity of proteins, relegated RNA to limited metabolic chores, as evolution and selection pressed populations into the extant world of DNA and proteins. The modern genetic code may have started as an operational RNA code or a primitive second genetic code. The codon-amino acid relationships were formed through interactions between residues and RNA molecules, perhaps out of aminoacylation reactions, that linked specific amino acids with specific RNAs. These RNAs may have been minihelix-like structures,

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