exploring the significance of initiation methionine by creation of non-methionine translation initiation E. coli

Research Project

Project/Area Number	18K19167
Research Category	Grant-in-Aid for Challenging Research (Exploratory)
Allocation Type	Multi-year Fund
Review Section	Medium-sized Section 38:Agricultural chemistry and related fields
Research Institution	The University of Tokyo
Principal Investigator	Nagao Asuteka 東京大学, 大学院工学系研究科(工学部), 助教 (30588017)
Project Period (FY)	2018-06-29 – 2020-03-31
Project Status	Completed (Fiscal Year 2019)
Budget Amount *help	¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000) Fiscal Year 2019: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000) Fiscal Year 2018: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Keywords	タンパク質合成 / tRNA / メチオニン / 翻訳開始 / 開始tRNA
Outline of Final Research Achievements	The fact that protein synthesis begins with methionine is one of universal facts in living cells. However, the reason has not been elucidated. In this study, we attempted to elucidate the significance and function of initial methionine in protein synthesis by modifying initiator tRNA to charge amino acid other than methionine and creating E. coli cells in which protein synthesis begins with non-methionine amino acids. As a result, although we succeeded to construct the assay system for our purpose, it was found that the replacement of initial methionine caused the cell death, suggesting the essentiality of initial methionine in protein synthesis. On the other hand, in the process of this research, we found that mutations at position 37 of initiator tRNA reduced translation efficiency of non-AUG start mRNA, indicating that A37 of initiator tRNA plays an important role in translation initiation.
Academic Significance and Societal Importance of the Research Achievements	細胞内の全開始tRNAを目的の改変型にするために、大腸菌ゲノムを改変しカウンターセレクション法によって変異型tRNA発現プラスミドと完全に置き換える方法を確立した。これまで変異tRNAを発現させる方法はあったが、完全に置換する方法はなく、目的tRNAが細胞内で機能するかどうかの判別に有効である。今回、目的の変異開始tRNAは獲得できず開始メチオニンの重要性を強調する結果となったが、その過程でこれまで報告がないような37位の塩基が翻訳開始に重要な役割を示唆することができた。また、タンパク質N末端解析の必要性から、合成中新生鎖の質量分析解析法といった新しい検出法を確立し、学会等で報告している。