Regulation of Translation Termination

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）的停止而不是意义密码子的反应。翻译终止需要两类多肽释放因子（RFs）：一类因子，密码子特异性的RFs（原核生物中的RF1和RF2；真核生物中的eRF1）；一类因子，非特异性的RFs（原核生物中的RF3；真核生物中的eRF3），它结合鸟嘌呤核苷酸并刺激一类RF活性。“释放因子tRNA模仿”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仿来读取遗传密码（停止密码子）。我们确定了RFs中有助于停止密码子识别的关键氨基酸（和基序）。首先，通过诱变细菌RF2（特异于UGA/UAA），然后选择温度敏感的RF1（特异于UAG/UAA）突变的抑制因子，分离出突变体。该突变体RF2在167位点携带Glu的单次替换Lys，并获得了除UGA和UAA外在UAG终止蛋白合成的ab…More能力，表明167位点及其附近通过在第三碱基（motif I）上的歧视直接参与终止密码子识别。其次，RF2的定点诱变突出了位置205、206、207和213的残基；它们的改变对细胞是有毒的，并诱导意义密码子的异常终止（基序II）。这些显性致死效应经常被基序I的突变所抑制，揭示了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进行转换。这些结果表明，RE蛋白编码一种蛋白。第四，拴链自由基足迹数据揭示了RE1预测的“反密码子-干模仿域”尖端接近30S解码位点，为提出的RF分子模仿提供了拓扑支持。与翻译的起始和延伸步骤类似，终止步骤包括由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位点的“ef - tu样”载体蛋白或“ef - g样”转位酶蛋白。我们获得的数据支持eRF3在真核生物中比EF-G在功能上更接近EF-Tu，而RF3在原核生物中比EF-Tu更接近EF-G。少

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