EVIDENCE FOR THE DOUBLE-SIEVE EDITING MECHANISM IN PROTEIN-SYNTHESIS - STERIC EXCLUSION OF ISOLEUCINE BY VALYL-TRANSFER RNA-SYNTHETASES
EVIDENCE FOR THE DOUBLE-SIEVE EDITING MECHANISM IN PROTEIN-SYNTHESIS - STERIC EXCLUSION OF ISOLEUCINE BY VALYL-TRANSFER RNA-SYNTHETASES
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DOI：
10.1021/bi00579a030
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发表时间：
1979-01-01
期刊：
影响因子：
2.9
通讯作者：
DINGWALL, C
中科院分区：
文献类型：
--
作者：
FERSHT, AR;DINGWALL, C
提出了一个简单的立体化学模型（双筛）的证据，通过该模型，在用正确的氨基酸填充 tRNA 的水平上保留了遗传编码过程的精确保真度。氨酰-tRNA 合成酶上的合成和水解（编辑）位点充当一对筛子，根据大小和化学性质对氨基酸进行粗略分类，从而实现氨基酸识别的高精度。合成位点在可接受的水平上拒绝大于特定底物的氨基酸；水解位点破坏小于特定底物（或与特定底物等排）的氨基酸的反应产物。通过绘制氨酰-tRNA合成酶催化的错误激活范围来测试假设的编辑机制，会受到非特异性氨基酸制剂中特定氨基酸的微量杂质的阻碍。给出了清除这些杂质的酶法。 3 种代表性缬氨酰-tRNA 合成酶的表观异亮氨酸依赖性 PPi 交换活性的至少 97% 可归因于残留的痕量缬氨酸。来自大肠杆菌的酶对异亮氨酸的选择性大于6倍。 104.结合已知体内异亮氨酸和缬氨酸的浓度比，误差率小于3倍。 10-6 是针对将异亮氨酸误激活为缬氨酸而计算的。因此，异亮氨酸在激活步骤中被筛出，并且 Ile-tRNAVal 的形成速率非常低，以至于不需要后续编辑。
Evidence is presented for a simple stereochemical model, the double sieve, by which the exquisite fidelity of the genetic coding process is preserved at the level of charging tRNA with the correct amino acid. The high accuracy in the recognition of amino acids is achieved by the synthetic and hydrolytic (editing) sites on an aminoacyl-tRNA synthetase functioning as a pair of sieves, crudely sorting the amino acids according to size and chemical nature. The synthetic site rejects, at a tolerable level, amino acids larger than the specific substrate; the hydrolytic site destroys the reaction products of the amino acids which are smaller than (or isosteric with) the specific substrate. Testing hypothetical editing mechanisms by mapping out the range of misactivations catalyzed by the aminoacyl-tRNA synthetases is hampered by trace impurities of the specific amino acid in preparations of the nonspecific amino acids. An enzymic method is given for scavenging these impurities. At least 97% of the apparent isoleucine-dependent PPi exchange activity of 3 representative valyl-tRNA synthetases is attributable to residual traces of valine. The selectivity of the enzyme from Escherichia coli against isoleucine is greater than 6 .times. 104. Combined with the known ratio of concentrations of isoleucine and valine in vivo, an error rate of less than 3 .times. 10-6 is calculated for the mistaken activation of isoleucine for valine. Isoleucine is thus sieved out at the activation step and the rate of formation of Ile-tRNAVal is so low as not to require subsequent editing.