Molecular mechanism of trans-translation mediated by tmRNA and its application

tmRNA介导的反式翻译的分子机制及其应用

• 批准号: 17380061
• 负责人: MUTO Akira
• 金额: $ 10.17万
• 依托单位: Hirosaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17380061/
• 关键词: tmRNA; trans-translation; Bacillus subtlis; SmpB; ribosome; Stress response; RNA; Escherichia coli; 胞子形成; 分子擬態; タグペプチド

a) Molecular mechanism of trans-translation mediated by tmRNA To investigate the functional structure of tmRNA in trans-translation reaction, we have developed an in vitro trans-translation system using E. coli extracts. Many mutant tmRNAs were constructed by in vitro mutagenesis, and tested their activities as tRNA, mRNA and rribosome binding. From the results, we could identify the structures and sequences in tmRNA required for each function. The effect of several antibiotics on the trans-translation was investigated: we found that paromomycin directly binds to tmRNA causing the frame-shift of translation of tag-peptides.b) Physiological roles of trans-translation We have constructed an in vivo system in which trans-translation products can be detected in Bacillus subtilis and E. coli. Using this system, several reaction products were isolated and identified by the N-terminal amino acid sequence analyses. The sites of trans-translation in some of the products were determined by MAS-spectrometry. It was shown that the trans-translation is involved in various cellular activities, such as catabolite repression, amino acid metabolism, stress responses, sporulation and so on.Endspore formation of Bacillus subtilis strain is significantly impaired by deleting the gene for tmRNA. The genetic and biochemical experimental results revealed that the synthesis of sigma-Kduring the late stage of sporulation process is significantly impaired in the mutant cells.

a）tmRNA介导的反式翻译的分子机制为了研究tmRNA在反式翻译反应中的功能结构，我们建立了一个体外反式翻译系统，利用E.大肠杆菌提取物。采用体外诱变的方法构建了多种突变体tmRNAs，并检测了它们的tRNA、mRNA和核糖体结合活性。从结果中，我们可以确定每个功能所需的tmRNA的结构和序列。研究了几种抗生素对转译的影响：我们发现巴龙霉素直接与tmRNA结合，引起标签肽翻译的移码。B）转译的生理作用我们构建了一个在枯草芽孢杆菌和大肠杆菌中检测转译产物的体内系统。杆菌使用该系统，几个反应产物被分离和鉴定的N-末端氨基酸序列分析。通过质谱法测定了一些产物中的反式翻译位点。研究表明，该基因的反式翻译参与了枯草芽孢杆菌的代谢产物阻遏、氨基酸代谢、应激反应和孢子形成等多种细胞活动，缺失tmRNA基因可显著抑制枯草芽孢杆菌的孢子形成。遗传学和生化实验结果表明，突变体细胞在产孢后期合成σ-K的能力明显减弱。

项目成果

Inhibition of GTPase activity of RsgA by novel antibiotics

新型抗生素抑制 RsgA GTPase 活性

• 发表时间: 2006
• 作者: Kimura;T.;Murakami;T.;Hashimoto;M.;Okuno;T.;Muto;A.;& Himeno;H.
• 通讯作者: H.

A functional interaction of SmpB with tmRNA for determination of the resuming point of trans-translation

• DOI: 10.1261/rna.604907
• 发表时间: 2007-10-01
• 期刊: RNA
• 影响因子: 4.5
• 作者: Konno, Takayuki;Kurita, Daisuke;Himeno, Hyouta
• 通讯作者: Himeno, Hyouta

Molecular mechanism of trans-translation

反式翻译的分子机制

• 发表时间: 2007
• 期刊: Biochemistry (Tokyo) 79
• 作者: Himeno;H.;Kurita;D.;Tatada;K.;Konno;T.;Hanawa;K.;Takemoto;C.;Kawazoe;M.;Yokoyama;S.;Nameki;N.;Kawi;G & Muto;A.
• 通讯作者: A.

アミノグリコシド系抗生物質によるトランストランスレーション開始点のシフト

氨基糖苷类抗生素引起的翻译起始点的转变

• 发表时间: 2005
• 作者: 今野貴之、高橋千治、栗田大輔、浅野クリスナ、三浦比佳理、武藤あき;ら、姫野俵太
• 通讯作者: ら、姫野俵太

細胞における蛋白質の一生-生成・成熟・輸送・管理・分解・病態-(翻訳と共役した蛋白質品質管理システム:トランスレーション)(小椋光、遠藤斗志也、森正敬、吉田賢右編)、

细胞内蛋白质的生命 - 产生、成熟、运输、管理、降解和病理学 - （蛋白质质量控​​制系统与翻译：翻译）（由小仓光、远藤敏哉、森正孝和吉田健介编辑），

• 发表时间: 2005
• 作者: 姫野俵太、武藤あき;ら
• 通讯作者: ら