In vitro conversion of a methionine to a glutamine-acceptor tRNA.
In vitro conversion of a methionine to a glutamine-acceptor tRNA.
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蛋氨酸在体外转化为谷氨酰胺受体 tRNA。
DOI：
10.1021/bi00346a043
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
H. Pelka
中科院分区：
文献类型：
--
作者：
L. H. Schulman;H. Pelka
大肠杆菌tRNAfMet的衍生物含有一个改变的反密码子序列，CUA，已在体外酶促合成。通过在用RNase A有限消化完整tRNAfMet中切除正常反密码子CAU，然后用T4 RNA连接酶和多核苷酸激酶将CUA序列插入反密码子环中来制备变体tRNA。改变的甲硫氨酸tRNA表现出极大的增强氨酰化的速率由氨酰-tRNA合成酶和氨酰化的速率由甲硫氨酰-tRNA合成酶大幅下降。由同源和非同源酶的充电反应的动力学参数的测量表明，修饰的tRNA是一个更好的受体谷氨酰胺比蛋氨酸。错装率类似于先前报道的含有反密码子CUA的色氨酸琥珀抑制tRNA，su+7 tRNATrp，其在体内和体外都被谷氨酰胺氨酰化[Yaniv，M.，Folk，W. R.，贝格，P.，& Soll，L.等（1974）J. Mol. 86，245-260; Yarus，M.，诺尔顿河E、& Soll，L.（1977）在Nucleic Acid-Protein Recognition（Vogel，H.，编辑）pp 391-408，Academic Press，纽约]。本研究结果提供了额外的证据表明，氨酰化的特异性由N-aminyl-tRNA合成酶是敏感的非同源tRNA的核苷酸序列的微小变化，并在中间位置的反密码子的尿苷参与识别的tRNA底物由这种酶。
A derivative of Escherichia coli tRNAfMet containing an altered anticodon sequence, CUA, has been enzymatically synthesized in vitro. The variant tRNA was prepared by excision of the normal anticodon, CAU, in a limited digestion of intact tRNAfMet with RNase A, followed by insertion of the CUA sequence into the anticodon loop with T4 RNA ligase and polynucleotide kinase. The altered methionine tRNA showed a large enhancement in the rate of aminoacylation by glutaminyl-tRNA synthetase and a large decrease in the rate of aminoacylation by methionyl-tRNA synthetase. Measurement of kinetic parameters for the charging reaction by the cognate and noncognate enzymes revealed that the modified tRNA is a better acceptor for glutamine than for methionine. The rate of mischarging is similar to that previously reported for a tryptophan amber suppressor tRNA containing the anticodon CUA, su+7 tRNATrp, which is aminoacylated with glutamine both in vivo and in vitro [Yaniv, M., Folk, W. R., Berg, P., & Soll, L. (1974) J. Mol. Biol. 86, 245-260; Yarus, M., Knowlton, R. E., & Soll, L. (1977) in Nucleic Acid-Protein Recognition (Vogel, H., Ed.) pp 391-408, Academic Press, New York]. The present results provide additional evidence that the specificity of aminoacylation by glutaminyl-tRNA synthetase is sensitive to small changes in the nucleotide sequence of noncognate tRNAs and that uridine in the middle position of the anticodon is involved in the recognition of tRNA substrates by this enzyme.