Experimental simulation on molecular evolution of RNA enzymes (ribozymes)

RNA酶（核酶）分子进化的实验模拟

• 批准号: 07458179
• 负责人: INOUE Tan
• 金额: $ 4.99万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458179/
• 关键词: RNA; ribozyme; enzyme; Tetrahymena; in vitro selection; 分子進化

We have been investigating the structure-function relationship of the Tetrahymena ribozyme that is the most extensively studied group I self-splicing intron. During the course of this project, two new stem-loop structures (P2.1 and P9.1) in the RNA are identified as the peripherals that associate together via long-range Watson-Crick basepairings. It also became clear that one of them functions as a trans-activator for the intron lacking this region. The activation presumably depends on an unusual A-C baseparing and the nucleotides located nearby.A new technique by that active RNAs can be selected among the modified ones with nemerous numbers of mutations was developed. The technique is based on the combined use of in vitro selection and color colony assay. By employig this technique, we identified adenosines that are located close to each other in an unusual loop region are resposible for the activation of the intron RNA.A project that employs error-prone PCR technique is also now in progress.On the basis of these findings, we are now attempting to build new artificial domains that facilitates tthe splicing reaction by the catalytic RNA.

我们一直在研究四膜虫核酶的结构-功能关系，它是研究最广泛的I组自剪接内含子。在这个项目的过程中，RNA中的两个新的茎环结构(P2.1和P9.1)被鉴定为通过远程Watson-Crick基线连接在一起的外围设备。这也变得很清楚，其中一个作为内含子的反式激活缺少这个区域。这种激活可能依赖于一个不寻常的A-C碱基对和位于附近的核苷酸。通过这种新技术，可以从突变数量极少的修饰RNA中选择活性RNA。这项技术是基于体外选择和彩色菌落试验的组合使用的。通过使用这项技术，我们确定了位于不寻常环区的腺苷与内含子RNA的激活有关。一个使用容易出错的PCR技术的项目也在进行中。在这些发现的基础上，我们现在正试图构建新的人工结构域，以促进催化RNA的剪接反应。

项目成果

S.Watanabe, G.Kawai, H.Mutou, K.Watanabe, T.Inoue and S.Yokoyama: "An RNA fragment consisting of the P7and P9.0stems and the 3'terminal guanosine of the Tetrahymena group I intron" Nucleic Acids Research. vol.24. 1337-1344 (1966)

S.Watanabe、G.Kawai、H.Mutou、K.Watanabe、T.Inoue 和 S.Yokoyama：“由 P7 和 P9.0 茎以及四膜虫 I 组内含子的 3 末端鸟苷组成的 RNA 片段”核酸

渡辺悟、河合剛太、武藤裕、渡辺公網、井上丹、横山茂之: "An RNA fragment consisting of the P7 and P9.0 stems and the 3'terminal guanosine of the Tetrahymena group I intron" Nucleic Acids Research. 24(7). 1337-1344 (1996)

Satoru Watanabe、Kota Kawai、Yutaka Muto、Koami Watanabe、Tan Inoue、Shigeyuki Yokoyama：“由 P7 和 P9.0 茎以及四膜虫 I 组内含子的 3 末端鸟苷组成的 RNA 片段”核酸研究 24（ 7). 1337-1344 (1996)。

井川善也,太田英男,白石英秋,井上丹: "Long-range interaction between P2.1 and P9.1 peripheral domains in the Tetrahymena ribozyme" Nucleic Acids Reserach. (印刷中). (1997)

Yoshiya Ikawa、Hideo Ota、Hideaki Shiraishi、Tan Inoue：“四膜虫核酶中 P2.1 和 P9.1 外围结构域之间的长程相互作用”核酸研究（1997 年出版）。

井上善也,白石英秋,井上丹: "Characterization of the newly constructed domaoins that replace P5abc within the Tetrahymena ribozyme" FEBS Letters. 394. 5-8 (1996)

Yoshiya Inoue、Eiaki Shiraishi、Tan Inoue：“在四膜虫核酶中取代 P5abc 的新构建的结构域的表征”FEBS Letters 394. 5-8 (1996)。

井上善也,内藤善一,白石英秋,井上丹: "Structure, function and mulecular evolution of Group I intron ribozyme" Nucleic Acids Symposium Series. 35. 195-196 (1996)

Yoshiya Inoue、Yoshiichi Naito、Hideaki Shiraishi、Tan Inoue：“I 类内含子核酶的结构、功能和分子进化”核酸研讨会系列 35. 195-196 (1996)。