Elucidation of the molecular mechanism by which the nascent chain of E. coli SecM cooperatively stabilizes translation arrest

2022 Fiscal Year Final Research Report

• Project/Area Number: 21K15433
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 49050:Bacteriology-related
• Research Institution: Osaka University
• Principal Investigator: TANZAWA Takehito 大阪大学, 蛋白質研究所, 特任研究員(常勤) (80899328)
• Project Period (FY): 2021-04-01 – 2023-03-31
• Keywords: タンパク質合成 / リボソーム / クライオ電子顕微鏡構造解析 / 翻訳アレスト / SecM

Outline of Final Research Achievements

The objective of this application study is to better understand the SecM translational arrest mechanism by focusing on the secM mRNA, which pauses protein synthesis on ribosome, and by elucidating the structure of the 70S ribosome-SecM nascent chain complex (SecM-RNC).
I isolated intact 70S ribosome from Escherichia coli, transcribed secM mRNA in vitro, and prepared the SecM-RNC in a commercial E. coli cell-free translation system. Sucrose density gradient centrifugation, negative staining electron microscopy, and cryo-electron microscopy were used to confirm the formation of SecM-RNC. It has been reported that the SecM arrest is caused by Pro-tRNA binding to the ribosomal A site, and I have conducted the single particle analysis of the 70S ribosome in complex with three tRNAs. Analysis of the nascent chain region is currently ongoing.

Free Research Field

構造生物学

Academic Significance and Societal Importance of the Research Achievements

オニールレポートの2050年問題に取り上げられているように，薬剤耐性菌感染症による死亡率上昇の危険度は高まる一方であり，新規・改良型抗生物質の開発が急務である．
本研究で注目する真正細菌リボソームは主要な創薬ターゲットであり，現存する抗生物質の実に7割以上がリボソームを標的とする．SecMは生命維持に必須なATP駆動型膜タンパク質輸送チャネル（SecA）の上流ペプチドでSecA翻訳を調節するため，SecM翻訳アレストの分子レベルでの理解は新規薬剤探索への応用が期待できる．またSecMアレスト配列は分子生物学研究で広く利用されており，本研究成果はより精密な実験系の提案にも寄与できると期待している．