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Regulation of Translation Termination

翻译终止的规定

基本信息

批准号：
08044196
负责人：
NAKAMURA Yoshikazu
金额：
$ 22.85万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08044196/
关键词：
protein synthesis translation termination stop codon polypeptide release factor molecular mimicry tRNA ribosome protein anticodon ペプチド鎖解難因子 tRMA リボソームテトラヒメナ遺伝子発現の転写後調節

项目摘要

Termination of protein synthesis takes place on the ribosomes as a response to a stop, rather than a sense, codon in the 'decoding' site (A site). Translation termination requires two classes of polypeptide release factors (RFs) : a class-I factor, codon-specific RFs (RF1 and RF2 in prokaryotes ; eRF1 in eukaryotes), and a class-II actor, non-specific RFs (RF3 in prokaryotes ; eRF3 in eukaryotes) that bind guanine nucleotides and stimulate class-I RF activity. The model of 'release factor tRNA mimicry' (Cell 87 : 147, 1996) explains how protein reads the genetic code (stop codon) by assuming a potential anticodon mimicry. We identified crucial amino acids (and motifs) of RFs that contribute to stop codon recognition. First, a mutant was isolated by mutagenizing bacterial RF2 (specific to UGA/UAA) and then selecting a suppressor of a temperature-sensitive RF1 (specific to UAG/UAA) mutation. This mutant RF2 carried a single substitution of Lys for Glu at position 167, and acquired the ab … More ility to terminate protein synthesis at UAG, in addition to UGA and UAA, showing that position 167 and its vicinity are directly involved in stop codon recognition by means of discrimination at the third-base (motif I). Second, site-directed mutagenesis of RF2 highlighted residues at positions 205, 206, 207 and 213 ; their alterations are toxic to cells and induce abnormal termination at the sense codon(s) (motif II). These dominant lethal effects are frequently suppressed by mutations in motif I, revealing the functional interaction between the two regions. Third, the specificity of RF1 and RF2 can be converted by swapping these two motifs. These results demonstrate that RE protein encodes a protein. Fourth, the tethered radical footprinting data reveal the proximity of the tip of the predicted 'anticodon-stem mimicry domain' of RE1 to the 30S decoding site, providing the topological support for the proposed molecular mimicry of RF.Analogous to the initiation and elongation steps of translation, the termination step involves hydrolysis of GTP to GDP by RF3 or eRF3. The model of RF-tRNA mimicry predicts that class-II GTP/GDP-binding proteins, RF3 and eRF3, may be an 'EF-Tu-like' vehicle protein to bring class-I proteins to the A site of the ribosome or an 'EF-G-like' translocase protein. We obtained the data to support that eRF3 is functionally closer to EF-Tu than EF-G in eukaryotes, while RF3 is closer to EF-G than EF-Tu in prokaryotes. Less

蛋白质合成的终止发生在核糖体上，作为对‘解码’位点(A位点)的终止密码子而不是正义密码子的反应。翻译终止需要两类多肽释放因子：一类是密码子特异的多肽释放因子(原核生物中的RF1和RF2；真核生物中的eRF1)；另一类是非特异性多肽释放因子(原核生物中的Rf3；真核生物中的eRF3)，它与鸟嘌呤核苷酸结合并刺激I类RF活性。释放因子tRNA模拟模型(Cell 87：147,1996)解释了蛋白质如何通过假设潜在的反密码子模拟来读取遗传密码(终止密码子)。我们确定了RFS的关键氨基酸(和基序)，这些氨基酸(和基序)有助于停止密码子识别。首先，通过诱变细菌RF2(针对UGA/UAA)，然后选择温度敏感的RF1(针对UAG/UAA)突变的抑制子来分离突变株。该突变体rf2在第167位携带一次赖氨酸取代谷氨酸，并获得了ab…除UGA和UAA外，在UAG终止蛋白质合成的可能性更大，这表明167位及其附近通过第三个碱基(基序I)的区分直接参与终止密码子的识别。第二，Rf2的定点突变突出显示在第205、206、207和213位残基；它们的改变对细胞有毒性，并导致有义密码子(S)(基序II)的异常终止。这些显性致死效应经常被基序I的突变所抑制，揭示了这两个区域之间的功能相互作用。第三，RF1和RF2的特异性可以通过交换这两个基序来转换。这些结果表明，RE蛋白编码一种蛋白质。第四，拴系自由基足迹数据揭示了预测的Re1‘反密码子-茎模拟结构域’的末端接近30S解码位点，为所提出的RF分子模拟提供了拓扑支持。与翻译的起始和延伸步骤类似，终止步骤涉及Rf3或eRF3将GTP水解为GDP。RF-tRNA模拟模型预测，II类GTP/GDP结合蛋白Rf3和eRF3可能是将I类蛋白带到核糖体A位的‘EF-Tu样’载体蛋白，或者是‘EF-G样’转位酶蛋白。我们得到的数据支持，在真核生物中，eRF3在功能上比EF-Tu更接近EF-Tu，而在原核生物中，Rf3比EF-Tu更接近EF-G。较少