Application of an effective post-labeling method using RNA methyltransferases and synthetic AdoMet-derivatives.

2011 Fiscal Year Final Research Report

• Project/Area Number: 21603018
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Chemical biology
• Research Institution: The Institute of Physical and Chemical Research
• Principal Investigator: BESSHO Yoshitaka 独立行政法人理化学研究所, 機能解析第2研究チーム, チームリーダー (70242815)
• Research Collaborator: KAWANO Yoshiaki 独立行政法人理化学研究所, 生命系放射光利用システム開発ユニット, 専任技師 (70321750) ; SAULIUS Klimasauskas Institute of Biotechnology, Vilnius University, LITHUANIA, Professor
• Project Period (FY): 2009 – 2011
• Keywords: 生物・生体工学 / バイオテクノロジー / 酵素反応 / タンパク質 / メチル基転位酵素 / tRNA / 人工補酵素 / S-アデノシルメチオニン

Research Abstract

We utilized a chemical biology approach to develop a site-specific RNA labeling method, in which nucleic acids can be covalently modified at nucleotide precision using engineered methyltransferases and synthetic analogs of their cofactor, AdoMet. By conjugating fluorescent probes to the extended group of the synthetic AdoMet, we successfully developed the core technologies to incorporate fluorescent labels into microcrystals of complexes of tRNA and its maturation enzymes, which cannot be observed by visible light, and the instrument to detect the fluorescent label incorporated into the sample crystal. A microcrystal X-ray diffraction method was developed, using the fluorescent microcrystals of the complex of the post-labeled tRNA and its maturation enzymes and the co-axial microscope installed on the micro-focus beamline BL32XU of the SPring-8 synchrotron.

Research Products

• [Journal Article] Substrate tRNA recognition mechanism of a multi-site-specific tRNA methyltransferase, Aquifex aeolicus Trm1, based on the X-ray crystal structure (2011)
  • Author(s): Awai T, Ochi A, Ihsanawati I, Sengoku T, Hirata A, Bessho Y, Yokoyama S, and Hori H.
  • Journal Title: J Biol Chem Volume: 286(40) Pages: 35236-35246
  • Peer Reviewed
• [Journal Article] Crystal structure of the bifunctional tRNA modification enzyme MnmC from Escherichia coli (2011)
  • Author(s): Kitamura A, Sengoku T, Nishimoto M, Yokoyama S, and Bessho Y
  • Journal Title: Protein Sci Volume: 20(7) Pages: 1105-1113
  • Peer Reviewed
• [Journal Article] Structure of an archaeal homologue of the bacterial Fmu/RsmB/RrmB rRNA cytosine5-methyltransferase (2010)
  • Author(s): Hikida Y, Kuratani M, Bessho Y, Sekine SI, and Yokoyama S
  • Journal Title: Acta Crystallogr Sect D Volume: 66(Pt12) Pages: 1301-1307
  • Peer Reviewed
• [Journal Article] Crystal structure of Methanocaldococcus jannaschii Trm4 complexed with sinefungin (2010)
  • Author(s): Kuratani M, Hirano M, Goto-Ito S, Itoh Y, Hikida Y, Nishimoto M, Sekine S, Bessho Y, Ito T, Grosjean H, and Yokoyama S
  • Journal Title: J Mol Biol Volume: 401(3) Pages: 323-333
  • Peer Reviewed
• [Presentation] SPring-8/XFELの新しい光に期待するタンパク質生合成システム・分子イメージング手法の開発 (2011)
  • Author(s): 別所義隆
  • Organizer: 第4回アストロバイオロジーワークショップ
  • Place of Presentation: 神戸
  • Year and Date: 2011-11-27
• [Presentation] 原始生命における核酸修飾と遺伝暗号 (2011)
  • Author(s): 別所義隆
  • Organizer: 第84回日本生化学会大会
  • Place of Presentation: 京都
  • Year and Date: 2011-09-21
• [Presentation] Multisite specific tRNA methyltransferase Trm1 from a hyper-thermophilic eubacterium, Aquifex aeolicus (2011)
  • Author(s): Takako Awai, Anna Ochi, Ihsanawati, Toru Sengoku, Satoshi Kimura, Takashi Yokogawa, Akira Hirata, Yoshitaka Bessho, Tsutomu Suzuki, Shigeyuki Yokoyama, Hiroyuki Hori
  • Organizer: Thermophiles 2011
  • Place of Presentation: Montana USA
  • Year and Date: 2011-09-11
• [Presentation] SPring-8/XFELの新しい光に期待する古細菌RNA/RNP分子イメージング手法の開発 (2011)
  • Author(s): 別所義隆
  • Organizer: 第24回日本Archaea研究会講演会
  • Place of Presentation: 鶴岡
  • Year and Date: 2011-09-03
• [Presentation] Crystal structure of the bifunctional tRNA modification enzyme MnmC (2011)
  • Author(s): Aya Kitamura, Toru Sengoku, Madoka Nishimoto, Shigeyuki Yokoyama, Yoshitaka Bessho
  • Organizer: 16th Annual meeting of the RNA society(RNA2011)
  • Place of Presentation: Kyoto Japan
  • Year and Date: 2011-06-16
• [Presentation] 核酸化学修飾の役割と基本原材料 (2010)
  • Author(s): 別所義隆
  • Organizer: 第33回日本分子生物学会年会・第83回日本生化学会大会合同大会
  • Place of Presentation: 神戸
  • Year and Date: 2010-12-08
• [Presentation] The system-biological whole-cell project of a model organism, Thermus thermophilus HB8 (2010)
  • Author(s): Yoshitaka Bessho, Akeo Shinkai, and Seiki Kuramitsu
  • Organizer: 8th International Congress on Extremophiles(Extremophiles 2010)
  • Place of Presentation: Portugal
  • Year and Date: 2010-09-14
• [Presentation] ヒスチジルtRNA合成酵素のG(-1)塩基認識機構 (2009)
  • Author(s): 別所義隆、東島今日子、柴田理恵、西本まどか、倉光成紀、横山茂之
  • Organizer: 第11回R N Aミーティング
  • Place of Presentation: 新潟
  • Year and Date: 2009-07-28