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Nucleoside modification and evolution of the wobble rule

核苷修饰与摆动规则的演化

基本信息

批准号：
16510162
负责人：
TAKAI Kazuyuki
金额：
$ 2.05万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

In order to determine which hydrogen bonds are responsible for the binding of modified uridines at the first position of the anticodon to the guanosine at the third position of the codon, a method for introducing modified guanosines into a specific position of RNA molecules was explored as a first step. As a result, a method for enzymatic synthesis of 6-thioguanosine 5'-monophosphate was found. It was also found that inosine and 6-thioguanosine can be introduced into RNA molecules by transcription reactions.Then, a method for ligation of different RNA molecules for the preparation of translatable mRNA molecules was explored. Conventional methods with T4 DNA and RNA ligases had problems in yields and specificity. T4 RNA ligase 2, which was reported recently to catalyze ligation of double-stranded RNA, was usable for the ligation, but the products were not translated in a cell-free system from Escherichia coli. This may have been due to some unknown activity of the enzyme. On the other hand, an RNA ligase from wheat embryos was useful for linking RNA fragments in high yields. However, it was difficult to check if the 2'-phosphate, which could be removed if another enzyme worked well, was efficiently removed from the ligation product. This problem should be solved in future researches.In the course of the research, an X-ray crystallographic structure of the wobble base pair between a modified uridine and the guanosine was reported from a competing research group. The structure looked inconsistent with the hydrogen bonding pattern postulated in the working hypothesis of this project. However, it was possible, in our opinion, that the base pair observed in the crystal was most stable only under the condition of the crystallization, where the pH is lower than the physiological condition. Therefore, a manuscript was published, in which the physicochemical and biochemical backgrounds of our hypothesis were described thoroughly.

为了确定哪些氢键是导致反密码子第一个位置的修饰尿苷与密码子第三个位置的鸟苷结合的原因，我们首先探索了将修饰鸟苷引入RNA分子特定位置的方法。结果发现了酶法合成6-硫代鸟苷5′-单磷酸的方法。还发现肌苷和6-硫鸟苷可以通过转录反应引入RNA分子。然后，探索了一种将不同RNA分子连接在一起制备可翻译mRNA分子的方法。传统的T4 DNA和RNA连接酶方法在产率和特异性上存在问题。最近报道的催化双链RNA连接的T4 RNA连接酶2可用于连接，但产物不能在大肠杆菌的无细胞系统中翻译。这可能是由于某种未知的酶的活性。另一方面，来自小麦胚胎的RNA连接酶可用于高产量连接RNA片段。然而，很难检查是否有效地从结扎产物中去除2'-磷酸，如果另一种酶工作良好，可以去除2'-磷酸。这一问题需要在今后的研究中加以解决。在研究过程中，一个竞争研究小组报道了修饰的尿嘧啶和鸟苷之间的摆动碱基对的x射线晶体结构。该结构看起来与本项目工作假设中假设的氢键模式不一致。然而，在我们看来，有可能晶体中观察到的碱基对只有在pH低于生理条件的结晶条件下才最稳定。因此，我们发表了一篇论文，对我们的假设的物理化学和生物化学背景进行了全面的描述。