Language：English   Publishing type：Research paper (scientific journal)  

Reconstitution of the translation system including the full set of aminoacyl-tRNA synthetases (ARSs) has been achieved with the components from Escherichia coli and human. We are trying to reconstitute the plant translation system because plants are also essential targets of biotechnology. Some eukaryotic ARSs form multi-synthetase complexes (MSCs), while plant MSCs have not been fully characterized by the conventional top-down approaches isolating them from plant tissues. To reveal more about the plant MSCs by bottom-up approaches and to reconstitute the plant translation system, we attempted here to prepare individual wheat ARSs by E. coli expression methods and sequenced tRNAs expressed in wheat germs. The 16 ARSs other than CysRS, IleRS, LysRS and ThrRS were synthesized in E. coli and were purified to near homogeneity. Fourteen of them other than ValRS and AsnRS had their aminoacylation activity. Then, the 6 ARSs that were not prepared successfully in E. coli (ValRS, AsnRS, IleRS, ThrRS, LysRS, and CysRS) were synthesized in a wheat-germ cell-free translation system and purified. As a result, ThrRS and ValRS, but not the other four, could aminoacylate wheat germ tRNA. Some of the unsuccessful ARSs might require unidentified co-translational interactions for formation of their active forms. Finally, a reconstituted wheat translation system containing 8 ARSs, eEFs, eRFs, ribosome, and total tRNA was constructed. The system successfully translated an mRNA encoding a hemagglutinin-tag and a randomly generated sequence of 5 amino acids downstream of a cricket paralysis virus internal ribosome entry site, which enables translation independent of initiation factors.

DOI： 10.1016/j.biochi.2025.06.010

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Synthetic genes for the two subunits of phenylalanyl-tRNA synthetase (PheRS) from wheat were expressed in Escherichia coli. When each gene was induced individually, the α subunit with a cleavable 6 × His tag at the amino terminus was largely soluble, while the β subunit was almost completely insoluble. When the two subunits were co-expressed, a soluble fraction containing the two subunits were obtained. This was purified by a standard method in which the tag was cleaved off with a specific protease after affinity purification. As the sample contained slightly more PheRSα than PheRSβ, we further resolved the sample by gel filtration to obtain the fraction that showed the size of the conventional α2β2 tetrameric complex and contains an almost equal amount of the two subunits. The final yield was 0.6 mg per 1 liter of the culture medium, and the specific activity was 28 nmol min-1 mg-1, which was higher than that of a fraction purified from wheat germ. This recombinant PheRS was used, along with purified samples of the elongation factors and the ribosomes from wheat germ, for a poly(U)-dependent poly(Phe) synthesis reaction. The reaction was dependent on the added components and lasted for more than several hours.

DOI： 10.1080/10826068.2024.2324077

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Glycyl-tRNA synthetases (GlyRSs) have different oligomeric structures depending on the organisms. While a dimeric α2 GlyRS species is present in archaea, eukaryotes and some eubacteria, a heterotetrameric α2β2 GlyRS species is found in most eubacteria. Here, we present the crystal structure of heterotetrameric α2β2 GlyRS, consisting of the full-length α and β subunits, from Lactobacillus plantarum (LpGlyRS), gram-positive lactic bacteria. The α2β2LpGlyRS adopts the same X-shaped structure as the recently reported Escherichia coli α2β2 GlyRS. A tRNA docking model onto LpGlyRS suggests that the α and β subunits of LpGlyRS together recognize the L-shaped tRNA structure. The α and β subunits of LpGlyRS together interact with the 3'-end and the acceptor region of tRNAGly, and the C-terminal domain of the β subunit interacts with the anticodon region of tRNAGly. The biochemical analysis using tRNA variants showed that in addition to the previously defined determinants G1C72 and C2G71 base pairs, C35, C36 and U73 in eubacterial tRNAGly, the identification of bases at positions 4 and 69 in tRNAGly is required for efficient glycylation by LpGlyRS. In this case, the combination of a purine base at Position 4 and a pyrimidine base at Position 69 in tRNAGly is preferred.

DOI： 10.1093/jb/mvad043

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In almost all eubacteria, the AUA codon is translated by tRNAIle2 bearing lysidine at the wobble position. Lysidine is introduced by tRNAIle lysidine synthetase (TilS) via post-transcriptional modification of the cytidine of tRNAIle2 (CAU). Lactobacillus casei and Lactobacillus plantarum have tilS homologues and tRNAIle2 (CAU) genes. In addition, L. casei also has another tRNAIle2 gene with an UAU anticodon. L. plantarum has a tRNAIle (UAU)-like RNA. Here, we demonstrate that L. casei tRNAIle2 (UAU) is charged with isoleucine by L. casei isoleucyl-tRNA synthetase (IleRS) but not by L. plantarum IleRS, even though the amino acid identity of these two enzymes is over 60%. It has been reported that, in Mycoplasma mobile, which has its tRNAIle2 (UAU) but no tilS homologue, an Arg residue at position 865 of the IleRS is required for recognition of the UAU anticodon. This position is occupied by an Arg also in the IleRSs from both of the Lactobacillus species. Thus, other residues in L. casei, IleRS should also contribute to the recognition of tRNAIle2 (UAU). We found that a chimeric L. casei IleRS in which the N-terminal domain was replaced by the corresponding region of L. plantatarum IleRS has very low aminoacylation activity towards both tRNAIle2 (UAU) and tRNAIle1 (GAU). The A18G mutant had barely detectable aminoacylation activity towards either of the tRNAsIle . However, a double point mutant of A18G and G19N aminoacylated tRNAIle1 (GAU), but not tRNAIle2 (UAU). Our results suggest that, for L. casei IleRS, Ala18 and Gly19 also play a critical role in recognition of tRNAIle2 (UAU).

DOI： 10.1111/febs.16389

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