Structural and functional studies of multiple complexes of RNA polymerase

• Project/Area Number: 23370048
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: The Institute of Physical and Chemical Research (2012-2014); The University of Tokyo (2011)
• Principal Investigator: SEKINE Shun-ichi 独立行政法人理化学研究所, ライフサイエンス技術基盤研究センター, チームリーダー (50321774)
• Project Period (FY): 2011-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥19,890,000 (Direct Cost: ¥15,300,000、Indirect Cost: ¥4,590,000); Fiscal Year 2014: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2013: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2012: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2011: ¥6,890,000 (Direct Cost: ¥5,300,000、Indirect Cost: ¥1,590,000)
• Keywords: 転写 / RNAポリメラーゼ / 転写因子 / 校正 / Ｘ線結晶構造解析 / バクテリオファージ / 酵素 / クロスリンク / 構造機能相関 / X線結晶構造解

Outline of Final Research Achievements

RNA polymerase accomplishes transcription by temporally changing its conformation. We developed the CPX method to analyze the RNAP conformation in solution, and first established relationships between major transcription functions and RNAP’s tight-ratcheted structural forms. We also performed crystallographic analyses of RNAP complexes relevant to transcription initiation and elongation, and revealed the structural basis for proofreading, as well as the regulation mechanism by an external factor.

Research Products

• [Journal Article] The ratcheted and ratchetable structural States of RNA polymerase underlie multiple transcriptional functions. (2015)
  • Author(s): Sekine, S., Murayama, Y., Svetlov, V., Nudler, E., Yokoyama, S.
  • Journal Title: Mol. Cell Volume: 57 Issue: 3 Pages: 408-421
  • DOI: 10.1016/j.molcel.2014.12.014
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Time-resolved Raman and polyacrylamide gel electrophoresis observations of nucleotide incorporation and misincorporation in RNA within a bacterial RNA polymerase crystal. (2015)
  • Author(s): Antonopoulos, I.H., Murayama, Y., Warner, B.A., Sekine, S., Yokoyama, S., Carey, P.R.
  • Journal Title: Biochemistry Volume: 54 Issue: 3 Pages: 652-665
  • DOI: 10.1021/bi501166r
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Molecular basis of RNA polymerase promoter specificity switch revealed through studies of Thermus bacteriophage transcription regulator. (2014)
  • Author(s): Severinov, K,. Minakhin, L., Sekine, S., Lopatina, A., Yokoyama, S.
  • Journal Title: Bacteriophage Volume: 4
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris (2014)
  • Author(s): Toshiaki Higo, Noriyuki Suka, Haruhiko Ehara, Masatoshi Wakamori, Shin Sato, Hideaki Maeda, Shun-ichi Sekine, Takashi Umehara, and Shigeyuki Yokoyama
  • Journal Title: J Struct Funct Genomics Volume: 15 Issue: 4 Pages: 191-199
  • DOI: 10.1007/s10969-014-9190-1
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Structural basis for promoter specificity switching of RNA polymerase by a phage factor (2014)
  • Author(s): Tagami, S., Sekine, S., Minakhin, L., Esyunina, D., Akasaka, R., Shirouzu, M., Kulbachinskiy, A., Severinov, K. and Yokoyama, S.
  • Journal Title: Genes & Development Volume: 28 Issue: 5 Pages: 521-531
  • DOI: 10.1101/gad.233916.113
  • Peer Reviewed
• [Journal Article] Crystallization and preliminary X-ray crystallographic analyses of Thermus thermophilus backtracked RNA polymerase. (2013)
  • Author(s): Murayama, Y.
  • Journal Title: Acta Crystallogr. Sect. F Volume: 69 Issue: 2 Pages: 174-177
  • DOI: 10.1107/s1744309113000055
  • Peer Reviewed
• [Journal Article] Structural basis of transcription by bacterial and eukaryotic RNA polymerases. (2012)
  • Author(s): Sekine, S., et al.
  • Journal Title: Curr Opin Struct Biol. Volume: 22 Issue: 1 Pages: 110-118
  • DOI: 10.1016/j.sbi.2011.11.006
  • Peer Reviewed
• [Journal Article] A novel conformation of RNA polymerase sheds light on the mechanism of transcription (2011)
  • Author(s): Tagami, S., et al.
  • Journal Title: Transcription Volume: 2 Issue: 4 Pages: 162-167
  • DOI: 10.4161/trns.2.4.16148
  • Peer Reviewed
• [Journal Article] 転写因子によるRNAポリメラーゼの制御のメカニズム-細菌の転写を阻害する新たな仕組みの解明 (2011)
  • Author(s): 関根俊一, et al.
  • Journal Title: 化学と生物 Volume: 49 Pages: 732-734
• [Presentation] 新生RNAによるRNAポリメラーゼの構造変化と機能との相関 (2014)
  • Author(s): 関根俊一
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] R/T conformational switching of RNA polymerase for the multiple transcription functions. (2013)
  • Author(s): 関根俊一
  • Organizer: FASEB Science Research Conference, Mechanism & Regulation of Prokaryotic Transcription
  • Place of Presentation: Saxtons River, USA
• [Presentation] Structural basis for promoter switching of RNA polymerase by a phage-encoded factor (2013)
  • Author(s): 関根俊一
  • Organizer: FASEB Science Research Conference, Mechanism & Regulation of Prokaryotic Transcription
  • Place of Presentation: Saxtons River, USA
• [Presentation] Structural basis for promoter switching of RNA polymerase by a phage-encoded protein (2013)
  • Author(s): 関根俊一
  • Organizer: The 7th International Conference on Structural Genomics 2013 - Structural Life Science (ICSG2013-SLS)
  • Place of Presentation: 札幌 京王プラザホテル
• [Presentation] SMART: an S-S based method to detect RNA polymerase conformational change (2013)
  • Author(s): 関根俊一
  • Organizer: The 65th Fujihara Seminar International Symposium on Synthetic Biology of Unnatural Base Pairs and Amino Acids
  • Place of Presentation: 苫小牧 グランドホテルニュー王子
• [Presentation] 転写校正においてRNAポリメラーゼの構造と活性を切りかえるメカニズム (2012)
  • Author(s): 関根 俊一
  • Organizer: 第12回日本蛋白質科学会年会
  • Place of Presentation: 名古屋国際会議場
• [Presentation] 転写の校正反応においてRNAポリメラーゼの構造と活性を切りかえるメカニズム (2012)
  • Author(s): 関根 俊一
  • Organizer: 第35回日本分子生物学会年会
  • Place of Presentation: 福岡国際会議場
• [Book] ブルーバックス放射光が解き明かす驚異のナノ世界-魔法の光が拓く物質世界の可能性-「数万もの生命の設計図を間違いなく書き写せるのはなぜ?」 (2011)
  • Author(s): 日本放射光学会編
  • Total Pages: 276
  • Publisher: 講談社
• [Remarks] 理研ライフサイエンス技術基盤研究センター 超分子構造解析研究チーム
  • URL: http://www.clst.riken.jp/activity/structuralbiology_group1_team2.html
• [Remarks] 理研プレスリリース 「RNAポリメラーゼの働きを切り替えるメカニズムを解明」
  • URL: http://www.riken.jp/pr/press/2015/20150206_3/
• [Remarks] ライフサイエンス技術基盤研究センター 超分子構造解析研究チーム
  • URL: http://www.riken.go.jp/research/labs/clst/struct_synth_biol/struct_biol/supramol_struct_biol/
• [Remarks] 理化学研究所 ライフサイエンス技術基盤研究センター 超分子構造解析研究チーム
  • URL: http://www.riken.go.jp/research/labs/clst/struct_synth_biol/struct_biol/supramol_struct_biol/