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Dive into the research topics where Shuntaro Takahashi is active.

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Featured researches published by Shuntaro Takahashi.


Angewandte Chemie | 2013

Effect of Pressure on the Stability of G‐Quadruplex DNA: Thermodynamics under Crowding Conditions

Shuntaro Takahashi; Naoki Sugimoto

Under pressure: A DNA G-quadruplex was unfolded under high pressure, but crowding conditions repressed this effect owing to enthalpic contributions. Volumetric analysis showed that ethylene glycol or poly(ethylene glycol) decreased the volume change of the transition by more than fourfold owing to the alteration of the number and/or radii of hydrating water molecules.


Journal of the American Chemical Society | 2009

Real-time monitoring of cell-free translation on a quartz-crystal microbalance.

Shuntaro Takahashi; Masaaki Iida; Hiroyuki Furusawa; Yoshihiro Shimizu; Takuya Ueda; Yoshio Okahata

The efficiency of protein synthesis is often regulated post-transcriptionally by sequences within the mRNA. To investigate the reactions of protein translation, we established a system that allowed real-time monitoring of protein synthesis using a cell-free translation mixture and a 27 MHz quartz-crystal microbalance (QCM). Using an mRNA that encoded a fusion polypeptide comprising the streptavidin-binding peptide (SBP) tag, a portion of Protein D as a spacer, and the SecM arrest sequence, we could follow the binding of the SBP tag, while it was displayed on the 70S ribosome, to a streptavidin-modified QCM over time. Thus, we could follow a single turnover of protein synthesis as a change in mass. This approach allowed us to evaluate the effects of different antibiotics and mRNA sequences on the different steps of translation. From the results of this study, we have determined that both the formation of the initiation complex from the 70S ribosome, mRNA, and fMet-tRNA(fMet) and the accommodation of the second aminoacyl-tRNA to the initiation complex are rate-limiting steps in protein synthesis.


Journal of Biological Chemistry | 2008

Direct monitoring of allosteric recognition of type IIE restriction endonuclease EcoRII.

Shuntaro Takahashi; Hisao Matsuno; Hiroyuki Furusawa; Yoshio Okahata

EcoRII is a homodimer with two domains consisting of a DNA-binding N terminus and a catalytic C terminus and recognizes two specific sequences on DNA. It shows a relatively complicated cleavage reaction in bulk solution. After binding to either recognition site, EcoRII cleaves the other recognition site of the same DNA (cis-binding) strand and/or the recognition site of the other DNA (trans-binding) strand. Although it is difficult to separate these two reactions in bulk solution, we could simply obtain the binding and cleavage kinetics of only the cis-binding by following the frequency (mass) changes of a DNA-immobilized quartz-crystal microbalance (QCM) responding to the addition of EcoRII in aqueous solution. We obtained the maximum binding amounts (Δmmax), the dissociation constants (Kd), the binding and dissociation rate constants (kon and koff), and the catalytic cleavage reaction rate constants (kcat) for wild-type EcoRII, the N-terminal-truncated form (EcoRII N-domain), and the mutant derivatives in its C-terminal domain (K263A and R330A). It was determined from the kinetic analyses that the N-domain, which covers the catalytic C-domain in the absence of DNA, preferentially binds to the one DNA recognition site while transforming EcoRII into an active form allosterically, and then the secondary C-domain binds to and cleaves the other recognition site of the DNA strand.


Proceedings of the National Academy of Sciences of the United States of America | 2017

Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase

Shuntaro Takahashi; John A. Brazier; Naoki Sugimoto

Significance Alterations in nonduplex structures could play roles during DNA replication in the progression of cancer and other intractable diseases. These noncanonical structures differ topologically from each other. However, the role of these differences in diseases remains unknown. In this study, we found that the presence of i-motif structures in the template caused the DNA polymerase to stall immediately before these structures. The i-motif structures are more efficient than other structures, such as G-quadruplexes and hairpins, although their thermodynamic stabilities are similar. This inhibition effect of the DNA polymerase was regulated by molecular crowding, which mimics conditions in the cell. Therefore, it is possible that the i-motif could impede DNA replication or repair and cause genomic instability. Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases.


Molecules | 2013

Effect of Pressure on Thermal Stability of G-Quadruplex DNA and Double-Stranded DNA Structures

Shuntaro Takahashi; Naoki Sugimoto

Pressure is a thermodynamic parameter that can induce structural changes in biomolecules due to a volumetric decrease. Although most proteins are denatured by pressure over 100 MPa because they have the large cavities inside their structures, the double-stranded structure of DNA is stabilized or destabilized only marginally depending on the sequence and salt conditions. The thermal stability of the G-quadruplex DNA structure, an important non-canonical structure that likely impacts gene expression in cells, remarkably decreases with increasing pressure. Volumetric analysis revealed that human telomeric DNA changed by more than 50 cm3 mol−1 during the transition from a random coil to a quadruplex form. This value is approximately ten times larger than that for duplex DNA under similar conditions. The volumetric analysis also suggested that the formation of G-quadruplex DNA involves significant hydration changes. The presence of a cosolute such as poly(ethylene glycol) largely repressed the pressure effect on the stability of G-quadruplex due to alteration in stabilities of the interactions with hydrating water. This review discusses the importance of local perturbations of pressure on DNA structures involved in regulation of gene expression and highlights the potential for application of high-pressure chemistry in nucleic acid-based nanotechnology.


Journal of the American Chemical Society | 2012

Traveling Time of a translating ribosome along messenger RNA monitored directly on a quartz crystal microbalance.

Shuntaro Takahashi; Kentaro Tsuji; Takuya Ueda; Yoshio Okahata

During translation, the biosynthesis of polypeptides is dynamically regulated. The translation rate along messenger RNA (mRNA), which is dependent on the codon, structure, and sequence, is not always constant. However, methods for measuring the duration required for polypeptide elongation on an mRNA of interest have not been developed. In this work, we used a quartz crystal microbalance (QCM) technique to monitor mRNA translation in an Escherichia coli cell-free translation system in real time. This method permitted us to evaluate the translation of proteins of interest fused upstream of a streptavidin-binding peptide (SBP) fusion protein. The translation of mRNA encoding the SBP fusion protein alone was observed as a mass increase on a streptavidin-modified QCM plate. Addition of the protein of interest resulted in a delay in the mass change corresponding to the traveling time of the ribosome along the coding region of the protein of interest. With this technique, the lengths of coding sequences, codon usages, influences of unique sequences, and various protein-coding sequences were evaluated. The results showed that the traveling time of the translating ribosome depends on the length of the coding region translated but is also affected by the sequence itself. Differences in the time lags for various proteins imply that mRNA coding sequences may regulate gene expression.


Journal of the American Chemical Society | 2013

Translation Enhancer Improves the Ribosome Liberation from Translation Initiation

Shuntaro Takahashi; Hiroyuki Furusawa; Takuya Ueda; Yoshio Okahata

For translation initiation in bacteria, the Shine-Dalgarno (SD) and anti-SD sequence of the 30S subunit play key roles for specific interactions between ribosomes and mRNAs to determine the exact position of the translation initiation region. However, ribosomes also must dissociate from the translation initiation region to slide toward the downstream sequence during mRNA translation. Translation enhancers upstream of the SD sequences of mRNAs, which likely contribute to a direct interaction with ribosome protein S1, enhance the yields of protein biosynthesis. Nevertheless, the mechanism of the effect of translation enhancers to initiate the translation is still unknown. In this paper, we investigated the effects of the SD and enhancer sequences on the binding kinetics of the 30S ribosomal subunits to mRNAs and their translation efficiencies. mRNAs with both the SD and translation enhancers promoted the amount of protein synthesis but destabilized the interaction between the 30S subunit and mRNA by increasing the dissociation rate constant (koff) of the 30S subunit. Based on a model for kinetic parameters, a 16-fold translation efficiency could be achieved by introducing a tandem repeat of adenine sequences (A20) between the SD and translation enhancer sequences. Considering the results of this study, translation enhancers with an SD sequence regulate ribosomal liberation from translation initiation to determine the translation efficiency of the downstream coding region.


ChemBioChem | 2008

70 S ribosomes bind to Shine-Dalgarno sequences without required dissociations.

Shuntaro Takahashi; Ryoko Akita; Hisao Matsuno; Hiroyuki Furusawa; Yoshihiro Shimizu; Takuya Ueda; Yoshio Okahata

In all living systems, proteins are synthesized on ribosomes by translating sequences of mRNA. Translation processes are complex and highly regulated by various translation factors together with ribosomes. It is known that translation initiation is the most dynamic step in building an initiation complex composed of a ribosome, mRNA, and an initiator tRNA. 2] In bacteria, it is thought that the 70S ribosome must dissociate into a 30S and 50S subunit triggered by initiation factors to interact with mRNA. It is generally accepted that the free 30S ribosomal subunit binds to a Shine–Dalgarno (SD) sequence, a 3–10 nucleotide purine-rich sequence (for example, AGGAGG), on a mRNA in the first step of the initiation translation, and then the 50S subunit is recruited to the mRNA–30S complex with an initiator tRNA on an AUG start codon downstream of the SD sequence. On the other hand, the preassembled 70S ribosome can translate only a leaderless mRNA lacking the SD sequence with an AUG start codon at the 5’ terminus of the mRNA. 5] Although most free ribosomes exist in the 70S form under physiological conditions, very few studies have investigated the binding of 70S to a mRNA with a SD sequence. 5] The interACHTUNGTRENNUNGaction of the intact 70S ribosome with the SD sequence has either been overlooked or not established in the literature. In this communication, we report that the intact 70S ribosome can bind directly to mRNA with the SD and AUG sequences without a required dissociation. Binding kinetics were studied using a mRNA-immobilized 27 MHz quartz-crystal microACHTUNGTRENNUNGbalance (QCM; Figure 1), which is known to be a very sensitive mass measuring device in aqueous solutions. QCM resonance frequencies decrease linearly upon mass increases on the QCM electrode to the nanogram level. Calibrations of the 27 MHz QCM in aqueous solutions are described in the Supporting Information (S1), and 1 Hz of frequency decrease was calibrated as an increase in 0.18 ng cm 2 of ribosomes in aqueous solution. E. coli 70S ribosomes and 30S and 50S ribosomal subunits were prepared and purified as described previously. Initiator tRNAs (tRNA) were prepared from an overexpressed strain and purified. fMet-tRNA was enzymatically methionylated by methionyl-tRNA synthetase and then formylated by methionyl-tRNA formyltransferase. mRNAs were prepared by in vitro transcription using a T7 RNA polymerase. For biotinylation of the 3’ terminus of the mRNA, the transcript was oxidized with NaIO4 and then modified with biotin hydrazide. We prepared several kinds of biotinylated mRNAs with SD+ AUG or SD +UUG sequences, the mRNA without the SD sequence (non-SD), the mRNA in which the SD sequence is hybridized with the antisense DNA (anti-SD), and the mRNA in which a 5’untranslated region (5’-UTR) is hybridized with the antisense DNA (anti-UTR). The sequence of the transcripts are shown in Table 1, and preparation methods of mRNA and the sequence of antisense DNAs are described in the Supporting Information (S2 and S3). AFFINIX Q4 with four 500 mL cells equipped with a 27 MHz QCM plate at the bottom of each cell was used as a QCM instrument. Biotinylated mRNA was linked by avidin– biotin interactions on a QCM plate covered with NeutrAvidinB as previously reported. After immobilization of mRNA, ribosomes were injected into aqueous buffer (10 mm HEPES-KOH, pH 7.3, 100 mm NH4Cl, 5 mm MgACHTUNGTRENNUNG(OAc)2, 0.5 mm CaCl2, 25 8C) in a QCM cell. Experimental procedures are described in the Supporting Information (S4). Figure 2 A shows typical time courses of frequency decreases (mass increases) corresponding to additions of 30S, 50S, and 70S ribosomes in aqueous solutions. The 30S subunit showed relative binding to mRNA with SD and AUG sequences (SD+ AUG), but the 50S subunit showed little specific binding. The [a] Dr. S. Takahashi, R. Akita, Dr. H. Matsuno, Dr. H. Furusawa, Prof. Y. Okahata Frontier Collaborative Research Centre Department of Biomolecular Engineering, Tokyo Institute of Technology Nagatsuda, Midori-ku, Yokohama 226-8501 (Japan) Fax: (+81)45-924-5781 E-mail : [email protected] [b] Dr. Y. Shimizu, Prof. T. Ueda Department of Medical Genome Sciences Graduate School of Frontier Sciences, University of Tokyo FSB401, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562 (Japan) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Figure 1. Experimental setup for measuring the binding kinetics of ribosomes (70S, 50S, 30S, and crosslinked 70S) with/without fMet-tRNA to a biotinylated mRNA-NeutrAvidin-immobilized 27-MHz quartz-crystal microACHTUNGTRENNUNGbalance (QCM, AFFINIX Q4) in aqueous solutions.


Analytical Chemistry | 2016

Real-Time Monitoring of G-Quadruplex Formation during Transcription

Tamaki Endoh; Ambadas B. Rode; Shuntaro Takahashi; Yuka Kataoka; Masayasu Kuwahara; Naoki Sugimoto

Cotranscriptional folding of an RNA transcript enables formation of metastable RNA structures. Thermodynamic and kinetic properties of RNA G-quadruplex formation have previously been investigated using purified guanine-rich oligonucleotides. Here, we describe a method for analysis of cotranscriptional dynamics of the G-quadruplex formation based on real-time monitoring of the fluorescence of G-quadruplex ligands. For RNA sequences with the potential to form mutually exclusive hairpin or G-quadruplex structures, the efficiency of G-quadruplex formation during transcription depended on position of the hairpin forming sequence. The real-time monitoring enabled evaluation of environmental effects on RNA dynamics, as we demonstrated facilitation of post-transcriptional G-quadruplex formation under molecular crowding conditions. The strategy demonstrated here provides folding insights into the G-quadruplex during transcription that should be involved in gene regulation.


AMB Express | 2013

Monitoring and kinetic analysis of the molecular interactions by which a repressor protein, PhaR, binds to target DNAs and poly[(R)-3-hydroxybutyrate]

Miwa Yamada; Shuntaro Takahashi; Yoshio Okahata; Yoshiharu Doi; Keiji Numata

The repressor protein PhaR, which is a component of poly[(R)-3-hydroxybutyrate] granules, functions as a repressor of the gene expression of the phasin PhaP and of PhaR itself. We used a quartz crystal microbalance to investigate the binding behavior by which PhaR in Ralstonia eutropha H16 targets DNAs and amorphous poly[(R)-3-hydroxybutyrate] thin films. Binding rate constants, dissociation rate constants, and dissociation constants of the binding of PhaR to DNA and to amorphous poly[(R)-3-hydroxybutyrate] suggested that PhaR bind to both in a similar manner. On the basis of the binding rate constant values, we proposed that the phaP gene would be derepressed in harmony with the ratio of the concentration of the target DNA to the concentration of amorphous poly[(R)-3-hydroxybutyrate] at the start of poly[(R)-3-hydroxybutyrate] synthesis in R. eutropha H16.

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Yoshio Okahata

Tokyo Institute of Technology

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Hiroyuki Furusawa

Tokyo Institute of Technology

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Ryoko Akita

Tokyo Institute of Technology

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Ambadas B. Rode

Kongju National University

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