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Kimitsuna Watanabe

Publications and source records attributed to Kimitsuna Watanabe.

At least 37 records · Page 2Linked to original sources
PubMed2004

Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease.

Point mutations in the mitochondrial (mt) tRNA(Leu(UUR)) gene are responsible for mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), a subgroup of mitochondrial encephalomyopathic diseases. We previously showed that mt tRNA(Leu(UUR)) with an A3243G or T3271C mutation derived from patients with MELAS are deficient in a normal taurine-containing modification (taum5U; 5-taurinomethyluridine) at the anticodon wobble position. To examine decoding disorder of the mutant tRNA due to the wobble modification deficiency independent of the pathogenic point mutation itself, we used a molecular surgery technique to construct an mt tRNA(Leu(UUR)) molecule lacking the taurine modification but without the pathogenic mutation. This "operated" mt tRNA(Leu(UUR)) without the taurine modification showed severely reduced UUG translation but no decrease in UUA translation. We thus concluded that the UUG codon-specific translational defect of the mutant mt tRNAs(Leu(UUR)) is the primary cause of MELAS at the molecular level. This result could explain the complex I deficiency observed clinically in MELAS.

Base Sequence↗
PubMed2004

Characterization of the human mitochondrial methionyl-tRNA synthetase.

Human mitochondrial methionyl-tRNA synthetase (human mtMetRS) has been identified from the human EST database. The cDNA encodes a 593 amino acid protein with an 18 amino acid mitochondrial import signal sequence. Sequence analysis indicates that this protein contains the consensus motifs characteristic of a class I aminoacyl-tRNA synthetase but lacks the Zn(2+) binding motif and C-terminal dimerization region found in MetRSs from various organisms. The mature form of human mtMetRS has been cloned and expressed in Escherichia coli. Gel filtration experiments indicate that this protein functions as a monomer with an apparent molecular mass of 67 kDa. The kinetic parameters for activation of methionine have been determined for the purified enzyme. The K(M) and k(cat) for aminoacylation of E. coli initiator tRNA(f)(Met) are reported. The kinetics of aminoacylation of an in vitro transcript of human mitochondrial tRNA(Met) (mtRNA(Met)) have been determined. To address the effects of the modification of mtRNA on recognition of the mitochondrial tRNA by human mtMetRS, the kinetics of aminoacylation of native bovine mtRNA(Met) and of an in vitro transcript of the bovine mtRNA(Met) have also been investigated.

Acylation↗
PubMed2004

Crystallization and preliminary X-ray diffraction study of mammalian mitochondrial seryl-tRNA synthetase.

The mitochondrial seryl-tRNA synthetase (mt SerRS) from Bos taurus was overexpressed in Escherichia coli and crystallized using the sitting-drop vapour-diffusion method. Crystals grew in a very narrow range of conditions using PEG 8000 as precipitant at room temperature. An appropriate concentration of lithium sulfate was critical for crystal nucleation. Crystals diffracted well beyond a resolution of 1.6 A and were found to belong to the orthorhombic space group C222(1), with unit-cell parameters a = 79.89, b = 230.42, c = 135.60 A. There is one dimer (M(r) approximately 113 kDa) in the asymmetric unit, with a solvent content of 55%. Efforts to solve the phase problem by molecular replacement are under way.

Animals↗
PubMed2004

Structures of d(GCGAAGC) and d(GCGAAAGC) (tetragonal form): a switching of partners of the sheared G.A pairs to form a functional G.AxA.G crossing.

The DNA fragments d(GCGAAGC) and d(GCGAAAGC) are known to exhibit several extraordinary properties. Their crystal structures have been determined at 1.6 and 1.65 A resolution, respectively. Two heptamers aligned in an antiparallel fashion associate to form a duplex having molecular twofold symmetry. In the crystallographic asymmetric unit, there are three structurally identical duplexes. At both ends of each duplex, two Watson-Crick G.C pairs constitute the stem regions. In the central part, two sheared G.A pairs are crossed and stacked on each other, so that the stacked two guanine bases of the G.AxA.G crossing expose their Watson-Crick and major-groove sites into solvent, suggesting a functional role. The adenine moieties of the A(5) residues are inside the duplex, wedged between the A(4) and G(6) residues, but there are no partners for interactions. To close the open space on the counter strand, the duplex is strongly bent. In the asymmetric unit of the d(GCGAAAGC) crystal (tetragonal form), there is only one octamer chain. However, the two chains related by the crystallographic twofold symmetry associate to form an antiparallel duplex, similar to the base-intercalated duplex found in the hexagonal crystal form of the octamer. It is interesting to note that the significant difference between the present bulge-in structure of d(GCGAAGC) and the base-intercalated duplex of d(GCGAAAGC) can be ascribed to a switching of partners of the sheared G.A pairs.

Base Pairing↗
PubMed2004

Functional compatibility of elongation factors between mammalian mitochondrial and bacterial ribosomes: characterization of GTPase activity and translation elongation by hybrid ribosomes bearing heterologous L7/12 proteins.

The mammalian mitochondrial (mt) ribosome (mitoribosome) is a bacterial-type ribosome but has a highly protein-rich composition. Almost half of the rRNA contained in the bacterial ribosome is replaced with proteins in the mitoribosome. Escherichia coli elongation factor G (EF-G Ec) has no translocase activity on the mitoribosome but EF-G mt is functional on the E.coli ribosome. To investigate the functional equivalency of the mt and E.coli ribosomes, we prepared hybrid mt and E.coli ribosomes. The hybrid mitoribosome containing E.coli L7/12 (L7/12 Ec) instead of L7/12 mt clearly activated the GTPase of EF-G Ec and efficiently promoted its translocase activity in an in vitro translation system. Thus, the mitoribosome is functionally equivalent to the E.coli ribosome despite their distinct compositions. The mt EF-Tu-dependent translation activity of the E.coli ribosome was also clearly enhanced by replacing the C-terminal domain (CTD) of L7/12 Ec with the mt counterpart (the hybrid E.coli ribosome). This strongly indicates that the CTD of L7/12 is responsible for EF-Tu function. These results demonstrate that functional compatibility between elongation factors and the L7/12 protein in the ribosome governs its translational specificity.

Amino Acid Sequence↗
PubMed2004

Yeast Nfs1p is involved in thio-modification of both mitochondrial and cytoplasmic tRNAs.

The IscS protein is a pyridoxal phosphate-containing cysteine desulfurase involved in iron-sulfur cluster biogenesis. In prokaryotes, IscS is also involved in various metabolic functions, including thio-modification of tRNA. By contrast, the eukaryotic ortholog of IscS (Nfs1) has thus far been shown to be functional only in mitochondrial iron-sulfur cluster biogenesis. We demonstrate here that yeast Nfs1p is also required for the post-transcriptional thio-modification of both mitochondrial (mt) and cytoplasmic (cy) tRNAs in vivo. Depletion of Nfs1p resulted in an immediate impairment of the 2-thio-modification of 5-carboxymethylaminomethyl-2-thiouridine at the wobble positions of mt-tRNA(UUU)(Lys) and mt-tRNA(UUG)(Gln). In addition, we observed a severe reduction in the 2-thio-modification of 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) of cy-tRNA(UUU)(Lys2) and cy-tRNA(UUC)(Glu3), although the effect was somewhat delayed compared with that seen in mt-tRNAs. Mass spectrometry analysis revealed an increase in 5-methoxycarbonylmethyluridine concomitant with a decrease in mcm(5)s(2)U in cy-tRNAs that were prepared from Nfs1p-depleted cells. These results suggest that Nfs1p is involved in the 2-thio-modification of both 5-carboxymethylaminomethyl-2-thiouridine in mt-tRNAs and mcm(5)s(2)U in cy-tRNAs.

Blotting, Northern↗
PubMed2004

The pathogenic A4269G mutation in human mitochondrial tRNA(Ile) alters the T-stem structure and decreases the binding affinity for elongation factor Tu.

The A4269G mutation in the human mitochondrial (mt) tRNA(Ile) gene is associated with fatal cardiomyopathy. This mutation completely inhibits protein synthesis in mitochondria, thereby significantly reducing their respiratory activity. The steady-state amount of tRNA(Ile) in cells bearing the A4269G mutation is almost half that of control cells. We previously reported that this mutation causes tRNA(Ile) to be unstable both in vivo and in vitro. To investigate whether the instability of the mutant tRNA(Ile) is due to structural alterations, a nuclease-probing experiment was performed with a mitochondrial enzymatic extract, which showed that the A4269G mutation destabilizes the T-stem of the mutant tRNA(Ile). In addition, measurements of the binding affinity of the aminoacylated mutant tRNA(Ile) for mt elongation factor Tu (EF-Tu) showed that the mutant tRNA(Ile) binds mt EF-Tu less efficiently than the wild-type does. This observation provides insight into the molecular pathology associated with tRNA dysfunction caused by this pathogenic point mutation.

Base Sequence↗
PubMed2003

Structure of d(GCGAAAGC) (hexagonal form): a base-intercalated duplex as a stable structure.

A DNA fragment d(GCGAAAGC), postulated to adopt a stable mini-hairpin structure on the basis of its extraordinary properties, has been X-ray analyzed. Two octamers related by a crystallographic twofold symmetry are aligned in an antiparallel fashion and associate to form a duplex, which is maintained by two Watson-Crick G.C base pairs and a subsequent sheared G.A pair at both ends. The central two A residues are free from base-pair formation. The corresponding base moieties of the two strands are intercalated and stacked on each other, forming a long column of G(1)-C(2)-G(3)-A(4)-A(5)(*)-A(5)-A(4)(*)-G(3)(*)-C(2)(*)-G(1)(*) (asterisks indicate the counter-strand). The Watson-Crick and major-groove sites of the four stacked adenine bases are exposed to the solvent region, suggesting a functional role. Since this structural motif is similar to those found in the nonamers d(G(Br)CGAAAGCT) and d(G(I)CGAAAGCT), the base-intercalated duplex may be a stable form of the specific sequence. Electrophoresis results suggest that the octamer has two states, monomeric and dimeric, in solution depending on the Mg(2+) concentration. The present duplex is preferred under the crystallization conditions, which correspond to physiologically allowed conditions.

Crystallography, X-Ray↗
PubMed2003

Quick two-step RNA ligation employing periodate oxidation.

The introduction of modified or labeled nucleotides into RNA is a powerful RNA engineering tool as it enables us to investigate how native RNA modifications affect RNA function and structure. It also helps in the structural analysis of RNA. A modified nucleotide can be introduced into a specific position of RNA by the method of two-step enzymatic ligation of RNA fragments. However, this method requires a complicated purification step between the two ligation steps that results in low yields of the ligation product. Here we have developed a new ligation technique employing periodate oxide that eliminates this purification step. This increases the total yield of the ligation product and makes it a faster procedure.

Anticodon↗
PubMed2003

Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting mechanism.

In Leishmania tarentolae, all mitochondrial tRNAs are encoded in the nuclear genome and imported from the cytosol. It is known that tRNA(Glu)(UUC) and tRNA(Gln)(UUG) are localized in both cytosol and mitochondria. We investigated structural differences between affinity-isolated cytosolic (cy) and mitochondrial (mt) tRNAs for glutamate and glutamine by mass spectrometry. A unique modification difference in both tRNAs was identified at the anticodon wobble position: cy tRNAs have 5-methoxycarbonylmethyl-2- thiouridine (mcm(5)s(2)U), whereas mt tRNAs have 5- methoxycarbonylmethyl-2'-O-methyluridine (mcm(5)Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm(5)U) at its wobble position, which could represent a common modification intermediate for both modified uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria-specific tRNA(Lys)(UUU) from the cytosol and found mcm(5)U at its wobble position, while its mitochondrial counterpart has mcm(5)Um. Mt tRNA(Lys) and in vitro transcribed tRNA(Glu) were imported much more efficiently into isolated mitochondria than the native cy tRNA(Glu) in an in vitro importation experiment, indicating that cytosol-specific 2-thiolation could play an inhibitory role in tRNA import into mitochondria.

Animals↗
PubMed2003

An RNA-modifying enzyme that governs both the codon and amino acid specificities of isoleucine tRNA.

The AUA codon-specific isoleucine tRNA (tRNA(Ile)) in eubacteria has the posttranscriptionally modified nucleoside lysidine (L) at the wobble position of the anticodon (position 34). This modification is a lysine-containing cytidine derivative that converts both the codon specificity of tRNA(Ile) from AUG to AUA and its amino acid specificity from methionine to isoleucine. We identified an essential gene (tilS; tRNA(Ile)-lysidine synthetase) that is responsible for lysidine formation in both Bacillus subtilis and Escherichia coli. The recombinant enzyme complexed specifically with tRNA(Ile) and synthesized L by utilizing ATP and lysine as substrates. The lysidine synthesis of this enzyme was shown to directly convert the amino acid specificity of tRNA(Ile) from methionine to isoleucine in vitro. Partial inactivation of tilS in vivo resulted in an AUA codon-dependent translational defect, which supports the notion that TilS is an RNA-modifying enzyme that plays a critical role in the accurate decoding of genetic information.

Amino Acid Sequence↗
PubMed2003

Application of the RNA structure classification system, CSNA, to NMR structure determination.

CSNA is a computer system which classifies a set of RNA structures based on their structural characters; hydrogen bond and base-base stacking. CSNA has been applied to the RNA structure determination by NMR and it was found that CSNA could provide well converged groups as the lowest energy structures. Here, we further applied CSNA to the structure determination of a 31mer RNA forming a psuedoknot structure. It was demonstrated that CSNA is a useful tool for the RNA structure determination by NMR.

Base Sequence↗
PubMed2003

Decoding property of C5 uridine modification at the wobble position of tRNA anticodon.

Post-transcriptional modification at the first (wobble) position of the tRNA anticodon participates in precise decoding of the genetic code. We recently identified a novel taurine-containing modified uridine (tau m5U; 5-taurinomethyluridine) at the wobble position of mammalian mitochondrial tRNAs and found lack of this modification in mutant mitochondrial tRNAs from human pathogenic cells of the mitochondrial encephalomyopathies, investigate molecular pathogenesis of the diseases, decoding activity of wobble uridines with or without C5 modification was measured using E. coli cell-free translation system. It has been revealed that C5 modification has a functional role for stabilizing U:G wobble base pair.

Amino Acid Sequence↗
PubMed2003

Simple and rapid synthesis of siRNA derived from in vitro transcribed shRNA.

Temporal gene silencing in mammalian cells using small interfering RNA (siRNA) is an invaluable tool for mammalian genetics and is becoming established. However, systematic studies of siRNA such as large-scale target validations are limited due to the high cost of chemical synthesis of double-stranded RNAs. Here, we devise a simple, rapid, practical and cost-effective method for preparing active siRNA derived from short hairpin (sh) RNA which is transcribed from a single-stranded synthetic DNA template using T7 RNA polymerase. This method doesn't require any sequence-limitation in the selection of the target region of genes. We demonstrate efficient silencing of several genes by the transcribed siRNAs obtained by this method.

DNA-Directed RNA Polymerases↗
PubMed2003

Functional genetic selection of the decoding center in E. coli 16S rRNA.

Ribosomal RNAs (rRNAs) play crucial roles in protein biosynthesis. The decoding center of 16S rRNA in 30S subunit affords a place for interaction between mRNA and tRNA, and contributes to the fidelity of the decoding by monitoring the codon-anticodon base pairing. The helices 18 and 44 in 16S rRNA are known to be major components of the decoding center. To investigate functional role of the conserved sequence in rRNAs, we employed a new genetic method that allows us to identify and select from randomized E. coli rRNA libraries those rRNA sequences absolutely required for the ribosome function. Functional consensus sequences were identified in both helices, providing us with a new insight into the decoding mechanism.

Escherichia coli↗
PubMed2002

Characterization of the interaction between the nucleotide exchange factor EF-Ts from nematode mitochondria and elongation factor Tu.

Caenorhabditis elegans mitochondria have two elongation factor (EF)-Tu species, denoted EF-Tu1 and EF-Tu2. Recombinant nematode EF-Ts purified from Escherichia coli bound both of these molecules and also stimulated the translational activity of EF-Tu, indicating that the nematode EF-Ts homolog is a functional EF-Ts protein of mitochondria. Complexes formed by the interaction of nematode EF-Ts with EF-Tu1 and EF-Tu2 could be detected by native gel electrophoresis and purified by gel filtration. Although the nematode mitochondrial (mt) EF-Tu molecules are extremely unstable and easily form aggregates, native gel electrophoresis and gel filtration analysis revealed that EF-Tu.EF-Ts complexes are significantly more soluble. This indicates that nematode EF-Ts can be used to stabilize homologous EF-Tu molecules for experimental purposes. The EF-Ts bound to two eubacterial EF-Tu species (E.coli and Thermus thermophilus). Although the EF-Ts did not bind to bovine mt EF-Tu, it could bind to a chimeric nematode-bovine EF-Tu molecule containing domains 1 and 2 from bovine mt EF-Tu. Thus, the nematode EF-Ts appears to have a broad specificity for EF-Tu molecules from different species.

Animals↗
PubMed2002

Taurine as a constituent of mitochondrial tRNAs: new insights into the functions of taurine and human mitochondrial diseases.

Taurine (2-aminoethanesulphonic acid), a naturally occurring, sulfur-containing amino acid, is found at high concentrations in mammalian plasma and tissues. Although taurine is involved in a variety of processes in humans, it has never been found as a component of a protein or a nucleic acid, and its precise biochemical functions are not fully understood. Here, we report the identification of two novel taurine-containing modified uridines (5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine) in human and bovine mitochondrial tRNAs. Our work further revealed that these nucleosides are synthesized by the direct incorporation of taurine supplied to the medium. This is the first reported evidence that taurine is a constituent of biological macromolecules, unveiling the prospect of obtaining new insights into the functions and subcellular localization of this abundant amino acid. Since modification of these taurine-containing uridines has been found to be lacking in mutant mitochondrial tRNAs for Leu(UUR) and Lys from pathogenic cells of the mitochondrial encephalomyopathies MELAS and MERRF, respectively, our findings will considerably deepen our understanding of the molecular pathogenesis of mitochondrial encephalomyopathic diseases.

Anticodon↗