Faculty Profiles - MURAKAMI Hiroshi

Papers - MURAKAMI Hiroshi

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Leucyl/Phenylalanyl-tRNA-Protein Transferase-Mediated Chemoenzymatic Coupling of N-Terminal Arg/Lys Units in Post-translationally Processed Proteins with Non-natural Amino Acids

Masumi Taki, Atsushi Kuno, Shinsuke Matoba, Yuki Kobayashi, Junichiro Futami, Hiroshi Murakami, Hiroaki Suga, Kazunari Taira, Tsunemi Hasegawa, Masahiko Sisido

ChemBioChem Vol. 7 ( 11 ) page： 1676 - 1679 2006.11

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

The protein no longer terminates here. Non-natural amino acids were attached onto a tRNA and were then transferred with the aid of L/F-tRNA-protein transferase to the N termini of proteins possessing N-terminal Lys or Arg components. By this new technique, 1- and 2-naphthylalanine or 3-nitrotyrosine were coupled to the N termini of several proteins. A novel and convenient method for adding single Lys units at the N termini of various proteins was also developed.

DOI： 10.1002/cbic.200600181

Web of Science

PubMed

テクノ・トレンドフレキシザイム--遺伝暗号の拡張とリプログラミングへの応用

村上裕, 菅裕明

バイオテクノロジージャーナル Vol. 6 ( 6 ) page： 734-737 2006.11

A highly flexible tRNA acylation method for non-natural polypeptide synthesis

Hiroshi Murakami, Atsushi Ohta, Hiroshi Ashigai, Hiroaki Suga

Nature Methods Vol. 3 ( 5 ) page： 357 - 359 2006.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media {LLC}

Here we describe a de novo tRNA acytation system, the flexizyme (Fx) system, for the preparation of acyl tRNAs with nearly unlimited selection of amino and hydroxy acids and tRNAs. The combination of the Fx system with an appropriate cell-free translation system allows us to readily perform mRNA-encoded synthesis of proteins and short polypeptides involving multiple non-natural amino acids.

DOI： 10.1038/nmeth877

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PubMed

セントラルドグマをつくりかえる (特集生命をデザインする合成生物学)

村上裕, 菅裕明, 平尾一郎

バイオニクス Vol. 3 ( 3 ) page： 34-39 2006.3

Flexizyme as a versatile tRNA acylation catalyst and the application for translation. Reviewed International journal

Murakami H, Ohta A, Goto Y, Sako Y, Suga H

Nucleic acids symposium series (2004) ( 50 ) page： 35-36 - 6 2006

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

Here we describe a de novo tRNA acylation system consisting of artificial ribozymes. This system, the flexizyme (Fx) system, allows for the preparation of acyl-tRNAs with almost no limitation on the use of a variety of amino/hydroxy acids and tRNAs. To demonstrate its utility, we have carried out protein synthesis involving site-specific incorporation of nonnatural amino and hydroxy acids via amber or programmed frame-shift suppressions. We have also demonstrated peptide synthesis involving multiple nonnatural amino acids via sense codon suppression. The combination of the Fx system and appropriate cell-free translation is a powerful and flexible tool for mRNA-encoded synthesis of nonnatural polypeptides.

DOI： 10.1093/nass/nrl018

PubMed

In vitro evolution of flexizymes that function under the conditions in translation system. Reviewed

Ohuchi M, Murakami H, Suga H

Nucleic acids symposium series (2004) ( 50 ) page： 299-300 2006

Translation initiation by using various N-acylaminoacyl tRNAs. Reviewed International journal

Goto Y, Ashigai H, Sako Y, Murakami H, Suga H

Nucleic acids symposium series (2004) ( 50 ) page： 293-294 - 4 2006

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

Bioactive peptides isolated from natural sources have diverse acyl groups on the N-terminus. It is difficult to synthesize these peptides in vitro translation system because ribosomal peptide synthesis generally limits the N-terminal group to be N-formylmethionine (fMet). To overcome this restriction, we developed a novel methodology for the ribosomal synthesis of peptides having various terminal N-acyl groups with desired amino acids. In this methodology, two technologies, Flexizyme system consisting of artificial ribozymes and a reconstitute E. coli cell-free translation system (PURE system), were used. First, an amino acid carrying a desired N-acyl group was charged onto an initiation tRNA by the Flexizyme system. The addition of this N-acyl-aminoacyl-tRNA (N-acyl-aa-tRNA) to the PURE system allowed us to initiate the peptide synthesis with the designated N-acyl-amino acid. By means of this methodology, the translation was exclusively initiated by various N-terminal acyl groups as well as amino acids without contamination of N-formylmethionine.

DOI： 10.1093/nass/nrl146

PubMed

Programmable ribozymes for mischarging tRNA with nonnatural amino acids and their applications to translation

Dimitrios Kourouklis, Hiroshi Murakami, Hiroaki Suga

Methods Vol. 36 ( 3 ) page： 239 - 244 2005.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier {BV}

Here we describe a novel technology that allows users to charge nonnatural amino acids onto any tRNA. This technology is based on a resin-immobilized ribozyme system, called Flexiresin. It enables users to readily and rapidly synthesize misacylated tRNAs with a wide variety of phenylalanine analogs. Since Flexiresin is reusable and little effort is necessary for regeneration, it is economical and convenient. Moreover, it can adapt to virtually any tRNA chosen by the user, and can therefore be applied to not only a single site mutation but also multiple sites with designated nonnatural amino acids when both the amber and programmed frame-shift mutations are utilized. The original ribozyme utilized for Flexiresin was artificially generated in vitro, and thus the technology in principle could be broadened from Phe analogues to essentially any amino acid. (c) 2005 Published by Elsevier Inc.

DOI： 10.1016/j.ymeth.2005.04.001

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PubMed

Designer Ribozymes: Programming the tRNA Specificity into Flexizyme

Krishna Ramaswamy, Hirohide Saito, Hiroshi Murakami, Kiyotaka Shiba, Hiroaki Suga

Journal of the American Chemical Society Vol. 126 ( 37 ) page： 11454 - 11455 2004.9

Position‐specific incorporation of dansylated non‐natural amino acids into streptavidin by using a four‐base codon Open Access

Takahiro Hohsaka, Norihito Muranaka, Chie Komiyama, Kinue Matsui, Satomi Takaura, Ryoji Abe, Hiroshi Murakami, Masahiko Sisido

FEBS Letters Vol. 560 ( 1-3 ) page： 173 - 177 2004.2

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

<jats:p>Novel non‐natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four‐base codon in an <jats:italic>Escherichia coli</jats:italic> in vitro translation system. 2,6‐Dansyl‐aminophenylalanine (2,6‐dnsAF) was found to be incorporated into the protein more efficiently than 1,5‐dansyl‐lysine, 2,6‐dansyl‐lysine, and 1,5‐dansyl‐aminophenylalanine. Fluorescence measurements indicate that the position‐specific incorporation of the 2,6‐dnsAF is a useful technique to probe protein structures. These results also indicate that well‐designed non‐natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.</jats:p>

DOI： 10.1016/s0014-5793(04)00099-7

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PubMed

Using a Solid-Phase Ribozyme Aminoacylation System to Reprogram the Genetic Code Open Access

Hiroshi Murakami, Dimitrios Kourouklis, Hiroaki Suga

Chemistry & Biology Vol. 10 ( 11 ) page： 1077 - 1084 2003.11

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier {BV}

Here, we report a simple and economical tRNA aminoacylation system based upon a resin-immobilized ribozyme, referred to as Flexiresin. This catalytic system features a broad spectrum of activities toward various phenylalanine (Phe) analogs and suppressor tRNAs. Most importantly, it allows users to perform the tRNA aminoacylation reaction and isolate the aminoacylated tRNAs in a few hours. We coupled the Flexiresin system with a high-performance cell-free translation system and demonstrated protein mutagenesis with seven different Phe analogs in parallel. Thus, the technology developed herein provides a new tool that significantly simplifies the procedures for the synthesis of aminoacyl-tRNAs charged with nonnatural amino acids, which makes the nonnatural amino acid mutagenesis of proteins more user accessible.

DOI： 10.1016/j.chembiol.2003.10.010

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PubMed

[Artificial ribozymes: selection and application for nonnatural amino acid mutagenesis]. Reviewed

Murakami H, Suga H

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme Vol. 48 ( 11 Suppl ) page： 1511-1518 2003.8

人工リボザイム--試験管内分子進化と非天然アミノ酸変異法への応用 (化学と生物学の接点がつくるNewバイオテクノロジー) -- (新規遺伝子と蛋白質を創製する)

村上裕, 菅裕明

蛋白質核酸酵素 Vol. 48 ( 11 ) page： 1511-1518 2003.8

A Versatile tRNA Aminoacylation Catalyst Based on RNA Open Access

Hiroshi Murakami, Hirohide Saito, Hiroaki Suga

Chemistry & Biology Vol. 10 ( 7 ) page： 655 - 662 2003.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier {BV}

Aminoacyl-tRNA synthetase (ARS) ribozymes have potential to develop a novel genetic coding system. Although we have previously isolated such a ribozyme that recognized aromatic amino acids, it could not be used as a versatile catalyst due to its limited ability of aminoacylation to a particular tRNA used for the selection. To overcome this limitation, we used a combination of evolutionary and engineering approaches to generate an optimized ribozyme. The ribozyme, consisting of 45 nucleotides, displays a broad spectrum of activity toward various tRNAs. Most significantly, this ribozyme is able to exhibit multiple turnover activity and charge parasubstituted Phe analogs onto an engineered suppressor tRNA (tRNA(CCCG)(Asn)). Thus, it provides a useful and flexible tool for the custom synthesis of mischarged tRNAs with natural and non-natural amino acids.

DOI： 10.1016/s1074-5521(03)00145-5

Web of Science

PubMed

Random insertion and deletion mutagenesis. Reviewed

Murakami H, Hohsaka T, Sisido M

Methods in molecular biology (Clifton, N.J.) Vol. 231 page： 53 - 64 2003

Position-Specific Incorporation of a Fluorophore−Quencher Pair into a Single Streptavidin through Orthogonal Four-Base Codon/Anticodon Pairs

Masumi Taki, Takahiro Hohsaka, Hiroshi Murakami, Kazunari Taira, Masahiko Sisido

Journal of the American Chemical Society Vol. 124 ( 49 ) page： 14586 - 14590 2002.12

Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society ({ACS})

Four-base codon strategy was applied to incorporate a fluorophore-quencher pair into specific positions on a single protein; beta-anthraniloyl-L-alpha,beta-diaminopropionic acid (atnDap) was employed as a fluorophore and p-nitrophenylalanine (ntrPhe) as a quencher. Their positions were directed by the CGGG/CCCG and GGGC/CCCG four-base codon/anticoclon pairs and two doubly mutated streptavidins, i.e., ((52)atnDap, (84)ntrPhe) and ((54)ntrPhe, (84)atnDap) mutants were synthesized through Escherichia coli in vitro protein synthesizing systems. Intramolecular photoinduced electron transfer (ET) was observed as the decrease of intensity in steady-state fluorescence spectroscopy and as the shortening of fluorescence decaytimes. The quenching data indicated that the ET rate reflects the detailed structure of the protein.

DOI： 10.1021/ja017714+

DOI： 10.1021/ja017714

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PubMed

Aminoacyl-tRNA Synthesis by a Resin-Immobilized Ribozyme

Hiroshi Murakami, Neil J. Bonzagni, Hiroaki Suga

Journal of the American Chemical Society Vol. 124 ( 24 ) page： 6834 - 6835 2002.6

Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs

Hiroshi Murakami, Takahiro Hohsaka, Masahiko Sisido

Nature Biotechnology Vol. 20 ( 1 ) page： 76 - 81 2002.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media {LLC}

A general method was developed for the construction of a library of mutant genes. The method, termed random insertion/deletion (RID) mutagenesis, enables deletion of an arbitrary number of consecutive bases at random positions and, at the same time, insertion of a specific sequence or random sequences of an arbitrary number into the same position. The applicability of the RID mutagenesis was demonstrated by replacing three randomly selected consecutive bases by the Bg/ll recognition sequence (AGATCT) in the GFP(uv) gene. In addition, the randomly selected three bases were replaced by a mixture of 20 codons. These mutants were expressed in Escherichia coli, and those that showed fluorescence properties different from the wild-type GFP were selected. A yellow fluorescent protein and an enhanced green fluorescent protein, neither of which could be obtained by error-prone PCR mutagenesis, were found among the six mutants selected. Several mutants of the DsRed protein that show different fluorescence properties were also obtained.

DOI： 10.1038/nbt0102-76

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PubMed

A novel fluorescent nonnatural amino acid that can be incorporated into a specific position of streptavidin. Reviewed

Taki M, Hohsaka T, Murakami H, Kuno A, Hasegawa T, Taira K, Sisido M

Nucleic acids research. Supplement (2001) ( 2 ) page： 203 - 204 2002

Incorporation of Nonnatural Amino Acids into Proteins by Using Various Four-Base Codons in an <i>Escherichia coli</i> in Vitro Translation System

Takahiro Hohsaka, Yuki Ashizuka, Hikaru Taira, Hiroshi Murakami, Masahiko Sisido

Biochemistry Vol. 40 ( 37 ) page： 11060 - 11064 2001.9

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