• BMB Reports
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• v.31 no.6
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• pp.525-535
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• 1998

A critical step in the faithful translation of genetic information is specific tRNA recognition by aminoacyl-tRNA synthetases. These enzymes catalyze the covalent attachment of particular amino acids to the terminal adenosine of cognate tRNA substrates. In general, there is one synthetase for each of the twenty amino acids and each enzyme must discriminate against all of the cellular tRNAs that are specific for the nineteen noncognate amino acids. Primary sequence information combined with structural data have resulted in the division of the twenty synthetases into two classes. In recent years, several high-resolution co-crystal structures along with biochemical data have led to an increased understanding of tRNA recognition by synthetases of both classes. The anticodon sequence and the amino acid acceptor stem are the most common locations for critical recognition elements. This review will focus on acceptor stem discrimination by class II synthetases. In particular, the results of in vitro aminoacylation assays and site-directed and atomic group mutagenesis studies will be discussed. These studies have revealed that even subtle atomic determinants can provide signals for specific tRNA aminoacylation.

• 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.

• 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.

• Proceedings of the Korea Crystallographic Association Conference
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• 2003.05a
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• pp.17-17
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• 2003

tRNA (m¹ G37) methyltransferase (TrmD) catalyze s the trans for of a methyl group from S-adenosyl-L-methionine (AdoMet) to G/sup 37/ within a subset of bacterial tRNA species, which have a residue G at 36th position. The modified guanosine is adjacent to and 3' of the anticodon and is essential for the maintenance of the correct reading frame during translation. We have determined the first crystal structure of TrmD from Haemophilus influenzae, as a binary complex with either AdoMet or S-adenosyl-L-homocysteine (AdoHcy), as a ternary complex with AdoHcy/phosphate, and as an apo form. The structure indicates that TrmD functions as a dimer (Figure 1). It also suggests the binding mode of G/sup 36/G/sup 37/ in the active site of TrmD and catalytic mechanism. The N-terminal domain has a trefoil knot, in which AdoMet or AdoHcy is bound in a novel, bent conformation. The C-terminal domain shows a structural similarity to DNA binding domain of trp or tot repressor. We propose a plausible model for the TrmD₂-tRNA₂ complex, which provides insights into recognition of the general tRNA structure by TrmD (Figure 2).

• Korean Journal of Microbiology
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• v.52 no.4
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• pp.406-414
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• 2016

Among 6 leu codons, CUG is the most frequently used codon in E. coli. It is recognized by leu-tRNA(CAG) encoded by four genes scattered on two chromosomal loci (leuT and leuPQV ). In the process of constructing a strain with no functional leu-tRNA (CAG) gene on chromosome, we made two mutant strains separately, one on leuPQV locus (${\Delta}leuPQV$), and the other on leuT locus [$leuT^*$(GAG)], where the anticodon of leuT was changed from CAG to GAG, thereby altering its recognition codon from CUG to CUC. We attempted to combine these two mutations by transduction using $leuT^*$(GAG) strain as a donor and ${\Delta}leuPQV$ strain as a recipient. Large and small colonies appeared from this transduction. From PCR and DNA sequencing, large colony was confirmed to be the reciprocal recombinant as expected, but the small colonies contained both mutant $leuT^*$(GAG) and wild type leuT (CAG) genes in the cell. This heterozygous diploid strain did not show any unusual morphology under microscopic observation, but, interestingly, it showed a linear growth curve in rich medium with much slower growth rate than wild type cell. It always formed homogenous small colonies in the selection medium, but, when there was no selection, it readily segregated into $leuT^*$(GAG) and leuT (CAG). From these observations, we suggested that the strain with both $leuT^*$(GAG) and leuT (CAG) genes was not a partial diploid (merodiploid), but a full diploid cell having two different chromosomes. We proposed a model explaining how such a heterozygous diploid cell was formed and how and why its growth showed a linear growth curve.