Functional Replacement of Hamster Lysyl-tRNA Synthetase by the Yeast Enzyme Requires Cognate Amino Acid Sequences for Proper tRNA Recognition - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Biochemistry Année : 1996

Functional Replacement of Hamster Lysyl-tRNA Synthetase by the Yeast Enzyme Requires Cognate Amino Acid Sequences for Proper tRNA Recognition

Résumé

We cloned the cDNA encoding a 597-aa hamster lysyl-tRNA synthetase. This enzyme is a close homologue of the 591-aa Saccharomyces cerevisiae enzyme, with the noticeable exception of their 60-aa N-terminal regions, which differ significantly. Several particular features of this polypeptide fragment from the hamster lysyl-tRNA synthetase suggest that it is implicated in the assembly of that enzyme within the multisynthetase complex. However, we show that this protein domain is dispensable in vivo to sustain growth of CHO cells. The cross-species complementation was investigated in the lysine system. The mammalian enzyme functionally replaces a null-allele of the yeast KRS1 gene. Conversely, the yeast enzyme cannot rescue Lys-101 cells, a CHO cell line with a temperature-sensitive lysyl-tRNA synthetase. The yeast and mammalian enzymes, overexpressed in yeast, were purified to homogeneity. The hamster lysyl-tRNA synthetase efficiently aminoacylates both mammalian and yeast tRNALys, whereas the yeast enzyme aminoacylates mammalian tRNALys with a catalytic efficiency 20-fold lower, as compared to its cognate tRNA. The 152-aa C-terminus extremity of the hamster enzyme provides the yeast enzyme with the capacity to complement Lys-101 cells. This hybrid protein is fairly stable and aminoacylates both yeast and mammalian tRNALys with similar catalytic efficiencies. Because this C-terminal polypeptide fragment is likely to make contacts with the acceptor stem of tRNALys, we conclude that it should carry the protein determinants conferring specific recognition of the cognate tRNA acceptor stem and therefore contributes an essential role in the operational RNA code for amino acids.

Dates et versions

hal-03276207 , version 1 (01-07-2021)

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Fabrice Agou, Sophie Quevillon, Pierre Kerjan, Marie-Thérèse Latreille, Marc Mirande. Functional Replacement of Hamster Lysyl-tRNA Synthetase by the Yeast Enzyme Requires Cognate Amino Acid Sequences for Proper tRNA Recognition. Biochemistry, 1996, 35 (48), pp.15322-15331. ⟨10.1021/bi9617926⟩. ⟨hal-03276207⟩
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