• Journal of the Korean Magnetic Resonance Society
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• v.20 no.2
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• pp.50-55
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• 2016

Backbone $^1H$, $^{13}C$ and $^{15}N$ resonance assignments are presented for the anticodon binding domain (residues 557-674) of human glycyl-tRNA synthetase (GRS). Role of the anticodon binding domain (ABD) of GRS as an anticancer ligand has recently been reported and its role in other diseases like Charcot-Marie-Tooth (CMT) and polymyositis have increased its interest. NMR assignments were completed using the isotope [$^{13}C/^{15}N$]-enriched protein and chemical shifts based secondary structure analysis with TALOS+ demonstrate similar secondary structure as reported in X-ray structure PDB 2ZT8, except some C-terminal residues. NMR signals from the N-terminal residues 557 to 571 and 590 to 614 showed very weak or no signals exhibiting dynamics or conformational exchange in NMR timescale.

• BMB Reports
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• v.28 no.4
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• pp.363-367
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• 1995

E. coli methionyl-tRNA synthetase is one of the class I tRNA synthetases. The Tryptophane residue at the position 461 located in the C-terminal domain of the enzyme is a key amino acid for the interaction with the anticodon of $tRNA^{Met}$. W461 was replaced with other amino acids to determine the chemical requirement for the interaction with the anticodon of $tRNA^{Met}$. Saturation mutagenesis at the position 461 generated a total of 12 substitution mutants of methionyl-tRNA synthetase. All the mutants showed the same in vivo stability as the wild-type enzyme, suggesting that the amino acid substitutions did not cause severe conformational change of the protein The mutants containing tyrosine, phenylalanine, histidine and cysteine substitutions showed in vivo activity while all the other mutants did not. The comparison of the in vitro aminoacylation activities of these mutants showed that aromatic ring structure, Van der Waals volume and hydrogen bond potential of the amino acid residue at the position 461 are the major determinants for the interaction with the anticodon of $tRNA^{Met}$.

• Bulletin of the Korean Chemical Society
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• v.4 no.3
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• pp.133-139
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• 1983

To understand the importance of Y-base adjacent to the anticodon stabilizing codon-anticodon interaction, a study has been undertaken for the model compound involving the interaction between Y-base and anticodon as well as the participation of water molecule by calculating the conformational free energy using an empirical potential function. We restrict our analysis to sites directly associated with Y-base by varying only the backbone torsion angles of Y-base. The hydration and $Mg^{+2}$ binding effects on the conformational stability of Y-base are calculated and discussed. The free Y-base is proved to be less stable than the hydrated one. The free energy change due to the hydration of Y-base amounts to -119.5 kcal/mole, in which the conformational energy change is -142.4 kcal/mole and the configurational entropy change is -76.9 e. u. It is found that the water molecules bound to Y-base and $Mg^{+2}$ contribute to the conformation of Y-base dominantly.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.101-108
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• 2000

The liverwort Pellia borealis is a diploid, monoecious, allopolypliod species (n=18) that as it was postulated, originated after hybridization and duplication of chromosome sets of two cryptic species: Pellia epiphylta-species N (n=9) and Pellia epiphylla-species 5 (n=9). Our recent results have supported the allopolyploid origin of P.borealis. We have shown that the nuclear genome of P.borealis consists of two nuclear genomes: one derived from P.epiphylla-species N and the other from P.epiphylla-species 5. In this paper we show the origin of chloroplast and mitochondrial genomes in an allopolyploid species P.borealis. To our knowledge there is no information concerning the way of mitochondria and chloroplast inheritance in Brophyta. Using an allopolyploid species of p. borealis as a model species we have decided to look into chloroplast and mitochondrial genomes of P.borealis, P.epiphylla-species N and P.epiphylla-species S for nucleotide sequences that would allow us to differentiate between both cryptic species and to identify the origin of organelle genomes in the alloploid species. We have amplified and sequenced a chloroplast $tRNA^{Leu}$ gene (anticodon UAA) containing an intron that has shown to be highly variable in a nucleotide sequence and used for plant population genetics. Unfortunately these sequences were identical in all three liverwort species tested. The analysis of the nucleotide sequence of chloroplast, an intron containing $tRNA^{Gly}$ (anticodon UCC) genes, gave expected results: the intron nucleotide sequence was identical in the case of both P.borealis and P.epiphyllaspecies N, while the sequence obtained from P.epiphyllasperies S was different in several nucleotide positions. These results were confirmed by the nucleotide sequence of another chloroplast molecular marker the chloroplast, an intron-contaning $tRNA^{Lys}$ gene (anticodon UUU). We have also sequenced mitochondrial, an intron-containing $tRNA^{Ser}$ gene (anticodon GCU) in all three liverwort species. In this case we found that, as in the case of the chloroplast genome, P.borealis mitochondrial genome was inherited from P.epiphylla-species N. On the basis of our results we claim that both organelle genomes of P.borealis derived from P.epiphylla-species N.

• Genomics & Informatics
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• v.12 no.2
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• pp.71-75
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• 2014

The tRNA structure contains conserved modifications that are responsible for its stability and are involved in the initiation and accuracy of the translation process. tRNA modification enzymes are prevalent in bacteria, archaea, and eukaryotes. tRNA Gm18 methyltransferase (TrmH) and tRNA $m^1G37$ methyltransferase (TrmD) are prevalent and essential enzymes in bacterial populations. TrmH involves itself in methylation process at the 2'-OH group of ribose at the 18th position of guanosine (G) in tRNAs. TrmD methylates the G residue next to the anticodon in selected tRNA subsets. Initially, $m^1G37$ modification was reported to take place on three conserved tRNA subsets ($tRNA^{Arg}$, $tRNA^{Leu}$, $tRNA^{Pro}$); later on, few archaea and eukaryotes organisms revealed that other tRNAs also have the $m^1G37$ modification. The present study reveals Gm18, $m^1G37$ modification, and positions of $m^1G$ that take place next to the anticodon in tRNA sequences. We selected extremophile organisms and attempted to retrieve the $m^1G$ and Gm18 modification bases in tRNA sequences. Results showed that the Gm18 modification G residue occurs in all tRNA subsets except three tRNAs ($tRNA^{Met}$, $tRNA^{Pro}$, $tRNA^{Val}$). Whereas the $m^1G37$ modification base G is formed only on $tRNA^{Arg}$, $tRNA^{Leu}$, $tRNA^{Pro}$, and $tRNA^{His}$, the rest of the tRNAs contain adenine (A) next to the anticodon. Thus, we hypothesize that Gm18 modification and $m^1G$ modification occur irrespective of a G residue in tRNAs.

• BMB Reports
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• v.28 no.6
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• pp.494-498
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• 1995

Maize mitochondrial tRNAs for isoleucine have been isolated using a putative $tRNA^{Ile}$ gene probe which has been previously isolated and characterized. It contains the 5'-CAT anticodon which would normally recognize the AUG methionine codon. The nucleotide sequence of one of these tRNAs has been partially determined, and contains a modified nucleotide at the first position of the anticodon. This type of posttranscriptional modification event could change the specificity of amino acid acceptance of a tRNA, unlike that deduced from the corresponding gene. An aminoacylation experiment also demonstrated that these purified tRNAs have isoleucine acceptance activity but no methionine-accepting activity.

• Bulletin of the Korean Chemical Society
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• v.2 no.2
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• pp.66-71
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• 1981

The effects of cation on tRNA have been theoretically investigated using the semiempirical potential energy functions. The binding of $Mg^{2+}$ to the model compound and the hydration scheme of the anticodon loop have been determined, and their stabilization energies produced by the introduction of magnesium pentahydrate and water molecules in the first hydration shell were calculated. The results indicate that magnesium pentahydrate is important for decreasing the flexibility of the anticodon loop and satisfying the large Y37 stereochemically during the protein synthesis. The effects of $Mg^{2+}$ on the hydration scheme were also investigated.

• Journal of the Korean Magnetic Resonance Society
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• v.9 no.2
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• pp.110-121
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• 2005

E.coli methionyl tRNA synthetase consist of 676 amino acids and plays a key role in initiation of protein synthesis. The native form of this enzyme is a homodimer, but the monomeric enzyme truncated approximately C-terminal 120 amino acids retains the full enzymatic activities. X-ray crystal structure of the active monomeric enzyme shows that it has two domains. The N-terminal domain is thought to be a binding site for acceptor stem of tRNA, ATP, and methionine. The C-terminal domain is mainly a-helical and makes an interaction with the anticodon of $tRNA^{Met}$. Especially it is suggested that the region of helix-loop-helix including the tryptophan residue at the position 461 may be the essential for the interaction with anticodon of $tRNA^{Met}$. In this work the structure and function of E. coli methionyl-tRNA synthetase was studied by spectroscopic method (NMR, CD, Fluorescence). The importance of tryptophan residue at the position 461 was investigated by fluorescence spectroscopy. Tryptophan 461 is expected to be an essential site for the interaction between E. coli methionyl-tRNA synthetase and E. coli $tRNA^{Met}$. Proton and heteonuclear 2-dimensional NMR spectroscopy were also used to elucidate the protein-tRNA interaction.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.83-89
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• 2001

Recent advances in the structural and molecular biology uncovered that a set of translation factors resembles a tRNA shape and, in one case, even mimics a tRNA function for deciphering the genetic ：ode. Nature must have evolved this 'art' of molecular mimicry between protein and ribonucleic acid using different protein architectures to fulfill the requirement of a ribosome 'machine'. Termination of protein synthesis takes place on the ribosomes as a response to a stop, rather than a sense, codon in the 'decoding' site (A site). Translation termination requires two classes of polypeptide release factors (RFs)： a class-I factor, codon-specific RFs (RFI and RF2 in prokaryotes; eRFI in eukaryotes), and a class-IT factor, non-specific RFs (RF3 in prokaryotes; eRF3 in eukaryotes) that bind guanine nucleotides and stimulate class-I RF activity. The underlying mechanism for translation termination represents a long-standing coding problem of considerable interest since it entails protein-RNA recognition instead of the well-understood codon-anticodon pairing during the mRNA-tRNA interaction. Molecular mimicry between protein and nucleic acid is a novel concept in biology, proposed in 1995 from three crystallographic discoveries, one, on protein-RNA mimicry, and the other two, on protein-DNA mimicry. Nyborg, Clark and colleagues have first described this concept when they solved the crystal structure of elongation factor EF- Tu：GTP：aminoacyl-tRNA ternary complex and found its overall structural similarity with another elongation factor EF-G including the resemblance of part of EF-G to the anticodon stem of tRNA (Nissen et al. 1995). Protein mimicry of DNA has been shown in the crystal structure of the uracil-DNA glycosylase-uracil glycosylase inhibitor protein complex (Mol et al. 1995; Savva and Pear 1995) as well as in the NMR structure of transcription factor TBP-TA $F_{II}$ 230 complex (Liu et al. 1998). Consistent with this discovery, functional mimicry of a major autoantigenic epitope of the human insulin receptor by RNA has been suggested (Doudna et al. 1995) but its nature of mimic is. still largely unknown. The milestone of functional mimicry between protein and nucleic acid has been achieved by the discovery of 'peptide anticodon' that deciphers stop codons in mRNA (Ito et al. 2000). It is surprising that it took 4 decades since the discovery of the genetic code to figure out the basic mechanisms behind the deciphering of its 64 codons.

• Korean Journal of Microbiology
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• v.54 no.4
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• pp.420-427
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• 2018

The orthogonal pair of Saccharomyces cerevisiae tyrosyl-tRNA synthetase (Sc YRS) and a variant of E. coli initiator tRNA, fMam $tRNA_{CUA}$ which recognizes the amber stop codon is an effective tool for site-specific incorporation of unnatural amino acids into the protein in E. coli. To evolve the amber suppression activity of the orthogonal pair, we generated a mutant library of Sc YRS by randomizing two amino acids at 320 and 321 which involve recognition of the first base of anticodon in fMam $tRNA_{CUA}$. Two positive clones are selected from the library screening with chloramphenicol resistance mediated by amber suppression. They showed growth resistance against high concentration of chloramphenicol and their $IC_{50}$ values were approximately 1.7~2.3 fold higher than the wild type YRS. In vivo amber suppression assay reveals that mutant YRS-3 (mYRS-3) clone containing amino acid substitutions of P320A and D321A showed 6.5-fold higher activity of amber suppression compared with the wild type. In addition, in vitro aminoacylation kinetics of mYRS-3 also showed approximately 7-fold higher activity than the wild type, and the enhancement was mainly due to the increase of tRNA binding affinity. These results demonstrate that optimization of anticodon recognition by engineered aminoacyl tRNA synthetase improves the efficiency of unnatural amino acid incorporation in response to nonsense codon.