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  • br Acknowledgements br Introduction Aminoacyl tRNA synthetas

    2022-05-13


    Acknowledgements
    Introduction Aminoacyl-tRNA synthetases catalyze the formation of an ester bond between an amino LY2109761 and the 3′ end of a tRNA in a two-step reaction. First, the amino acid is activated and an enzyme-bound adenylate intermediate is formed. Then, the amino acid is transferred to the 3′ end of the tRNA molecule. Aminoacyl-tRNA synthetases have to decode the genetic information into amino acids with high fidelity and protein–tRNA interactions play a key role in this process. Binding of a tRNA molecule to a synthetase is thought to proceed in two steps: an initial broad-specificity interaction between the tRNA and the synthetase is followed by a more precise recognition that involves conformational changes of both the synthetase and the cognate tRNA and contributes to tRNA specificity as non-specific tRNAs are normally less efficiently aminoacylated. Prokaryotic synthetases, which represent the elementary synthetase throughout evolution, are built of two major functional domains constituting the LY2109761 catalytic core: the catalytic domain contains the active site and interacts with the acceptor arm of tRNA; the second domain generally interacts with the anticodon arm.4., 5. Eukaryotic enzymes present a catalytic core closely related to the respective prokaryotic enzyme, but possess additional domains appended to the N or C-terminal end. In higher eukaryotes, polypeptide extensions are hydrophobic and participate in the formation of the multi-synthetase complex, or are cationic and interact non-specifically with tRNA to improve the catalytic efficiency of the enzyme. According to their primary structures, tRNA-interacting factors (tIF) can be divided into three major families. The first one is found in eukaryotic LysRS, AspRS and AsnRS.8., 9., 10., 11. A repeated domain is found in human MetRS, HisRS, TrpRS, GlyRS and in the linker region of glutamyl-prolyl-tRNA synthetase.12., 13., 14., 15. The last type of tIF (EMAPII-like domain) is appended to plant MetRS or human TyrRS and acts in cis of the catalytic domain of the synthetase. The EMAPII domain was first identified in human p43, a non-synthetase protein associated with the multi-synthetase complex, and is identical with a tumor-derived factor (endothelial monocyte-activating polypeptide II) that alters endothelial functions. The yeast protein Arc1p (aminoacyl-tRNA synthetase cofactor I), which makes a ternary complex with GluRS and MetRS, possesses a C-terminal domain homologous to EMAPII and acts as a tIF in trans of these two synthetases.20., 21. Methionyl-tRNA synthetases display an especially variable structural organization throughout evolution. The enzyme can be limited to the catalytic core as in Aquifex aeolicus, or may have an appended C-terminal domain involved in dimer formation as in Escherichia coli.22., 23. Plant and human MetRS possess C-terminal extensions with unrelated sequences. In both cases, this domain interacts non-specifically with tRNA, and acts in cis to the catalytic core to improve the catalytic efficiency of the enzyme in the aminoacylation reaction.15., 16. In yeast, Arc1p possesses a tRNA-binding domain at its C-terminal end. The N-terminal moiety of Arc1p interacts with the eukaryotic-specific N-terminal domains of GluRS and MetRS. Association with Arc1p provides a tIF in trans to these two enzymes which improves their catalytic efficiency.20., 24., 25. Arc1p is required for optimal growth and is essential for viability of yeast in the absence of the nuclear pore-associated protein Los1p involved in nuclear tRNA export. Because EMAPII-like domains are found appended to various proteins and are widely distributed among living organisms, we investigated the possibility of rescuing Arc1p-deficient yeast cells with fusion proteins containing EMAPII-like domains from different origins. Using MetRS from the rice Oriza sativa (OsMetRS), we showed that in the absence of its C-terminal tIF, the catalytic core of plant MetRS could not complement an arc1−mes1− yeast strain. We constructed a series of fusion proteins that tested the capacity of various EMAPII-like domains to act as functional cis-acting tRNA-binding domains in vivo. To further address the function of these EMAPII-like domains, we tested for the absolute requirement of their association in cis or in trans of the synthetases to achieve complementation. Our results establish that human p43 or yeast Arc1p are also able to sustain growth of yeast strains independently of their function of tIF for MetRS and GluRS. These data suggest that in addition to its role of tIF for two synthetases, Arc1p may be directly involved in sequestering tRNA in the cytoplasm of yeast.