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）中的终止密码子而不是有义密码子的响应。翻译终止需要两类多肽释放因子（RF）：I类因子，密码子特异性RF（原核生物中的RF 1和RF 2;真核生物中的eRF 1），和II类因子，结合鸟嘌呤核苷酸并刺激I类RF活性的非特异性RF（原核生物中的RF 3;真核生物中的eRF 3）。“释放因子tRNA模拟”的模型（Cell 87：147，1996）解释了蛋白质如何通过假定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RF的关键氨基酸（和基序），有助于终止密码子识别。首先，通过诱变细菌RF 2（对UGA/UAA特异性），然后选择温度敏感性RF 1（对UAG/UAA特异性）突变的抑制子，分离突变体。该突变体RF 2在第167位由Lys取代为Glu，并获得了ab ...更多信息 除了UGA和UAA之外，在UAG处终止蛋白质合成的能力，表明位置167及其附近通过在第三碱基（基序I）处的辨别直接参与终止密码子识别。第二，RF 2的定点诱变突出显示了位置205、206、207和213处的残基;它们的改变对细胞有毒，并诱导有义密码子（基序II）处的异常终止。这些显性的致死作用经常被基序I的突变抑制，揭示了两个区域之间的功能相互作用。第三，RF 1和RF 2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蛋白编码蛋白质。第四，系留自由基足迹数据揭示了RE 1的预测的“反密码子茎模拟域”的尖端与30 S解码位点的接近，为所提出的RF的分子模拟提供了拓扑支持。类似于翻译的起始和延伸步骤，终止步骤涉及由RF 3或eRF 3将GTP水解为GDP。RF-tRNA模拟的模型预测，II类GTP/GDP结合蛋白，RF 3和eRF 3，可能是一个'EF-Tu-like'载体蛋白，使I类蛋白质的核糖体或'EF-G-like'易位酶蛋白的A位点。我们获得的数据支持eRF 3在真核生物中比EF-G功能更接近EF-Tu，而在原核生物中RF 3比EF-Tu功能更接近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