Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. coli tRNAfMet by E. coli Met-tRNA synthetase.
Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. coli tRNAfMet by E. coli Met-tRNA synthetase.
复制标题
反密码子摆动位置的碱基取代抑制大肠杆菌 Met-tRNA 合成酶对大肠杆菌 tRNAfMet 的氨酰化。
DOI：
10.1093/nar/11.5.1439
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发表时间：
1983
影响因子：
14.9
通讯作者：
Susani,M
中科院分区：
文献类型：
--
作者：
Schulman,LH;Pelka,H;Susani,M
大肠杆菌tRNAfMet的衍生物含有单碱基取代的摆动位置的反密码子已在体外酶促合成。该过程包括通过用RNA酶A有限消化完整的tRNAfMet来切除正常的反密码子CAU。然后使用RNA连接酶将四个三核苷酸NAU中的每一个连接到5′半分子上，并随后连接3′和修饰的5′片段以再生反密码子环。用此方法合成含有反密码子CAU的完整tRNAfMet，得到的产物与天然tRNAfMet在被大肠杆菌氨酰化的能力方面没有区别。大肠杆菌甲硫氨酰-tRNA合成酶。在tRNAfMett的摆动位置上的任何其他核苷酸的取代都会严重损害合成酶识别tRNA的能力。在高浓度纯酶存在下测定甲硫氨酸的接受度，已经确定tRNAfMetis的AAU、GAU和UAU反密码子衍生物的氨酰化速率比含有C作为摆动碱基的天然或合成的tRNA慢4至5个数量级。此外，无活性的tRNA衍生物不能抑制正常tRNAfMet的氨酰化，表明它们与酶的结合很差。这些结果支持一个模型，涉及直接相互作用之间的Met-tRNA合成酶和C在摆动位置在氨酰化tRNAfMet。
Derivatives of E.coli tRNAfMetcontaining single base substitutions at the wobble position of the anticodon have been enzymatically synthesized in vitro. The procedure involves excision of the normal anticodon, CAU, by limited digestion of intact tRNAfMetwith RNase A. RNA ligase is then used to join each of four trinucleotides, NAU, to the 5′ half molecule and to subsequently link the 3′and modified 5′ fragments to regenerate the anticodon loop. Synthesis of intact tRNAfMetcontaining the anticodon CAU by this procedure yields a product which is indistinguishable from native tRNAfMetwith respect to its ability to be aminoacylated by E. coli methionyl-tRNA synthetase. Substitution of any other nucleotide at the wobble position of tRNAfMetdrastically impairs the ability of the synthetase to recognize the tRNA. Measurement of methionine acceptance in the presence of high concentrations of pure enzyme has established that the rate of aminoacylation of the AAU, GAU and UAU anticodon derivatives of tRNAfMetis four to five orders of magnitude slower than that of the native or synthesized tRNA containing C as the wobble base. In addition, the inactive tRNA derivatives fail to inhibit aminoacylation of normal tRNAfMet, indicating that they bind poorly to the enzyme. These results support a model involving direct interaction between Met-tRNA synthetase and the C in the wobble position during aminocylation of tRNAfMet.