Senger B, Auxilien S, Englisch U, Cramer F, Fasiolo F
Institut de Biologie Moléculaire et Cellulaire, UPR 9002 15, Rue René Descartes, F-67084 Strasbourg Cedex, France.
Biochemistry. 1997 Jul 8;36(27):8269-75. doi: 10.1021/bi970206l.
Earlier work by two independent groups has established the fact that anticodons GAU and LAU of Escherichia coli tRNAIle isoacceptors play a critical role in the tRNA identity. Yeast possesses two isoleucine transfer RNAs, a major one with anticodon IAU and a minor one with anticodon PsiAPsi which are derived from the post-transcriptional modification of AAU and UAU gene sequences, respectively. We present direct evidence which reveals that inosine is a positive determinant for yeast isoleucyl-tRNA synthetase. We also show that yeast tRNAMet with guanosine at the wobble position becomes aminoacylated with isoleucine while methionine acceptance is lost. As inosine and guanosine share the 6-keto and the N-1 hydrogen groups, this suggests that these hydrogen donor and acceptor groups are determinants for isoleucine specificity. The role of the minor tRNAIle anticodon pseudouridines in tRNA isoleucylation could not be tested directly but was deduced from a 40-fold decrease in the activity of the unmodified transcript. The presence of the NHCO structure in guanosine, inosine, pseudouridine, and lysidine suggests a unifying model of wobble base recognition by the yeast and E. coli isoleucyl-tRNA synthetase. In contrast to lysidine which switches the identity of the tRNA from methionine to isoleucine [Muramatsu, T., Nishikawa, K., Nemoto, F., Kuchino, Y., Nishimura, S., Miyazawa, T., & Yokoyama, S. (1988) Nature 336, 179-181], pseudouridine-34 does not modify the specificity of the yeast minor tRNAIle since U-34 is a strong negative determinant for yeast MetRS. Therefore, the major role of Psi-34 (in combination with Psi-36 or not) is likely in isoleucine AUA codon specificity and translational fidelity.
两个独立研究小组早期的工作已经证实，大肠杆菌异亮氨酸转运核糖核酸（tRNAIle）同功受体的反密码子GAU和LAU在tRNA识别中起关键作用。酵母有两种异亮氨酸转运核糖核酸，一种主要的反密码子为IAU，另一种次要的反密码子为PsiAPsi，它们分别来自AAU和UAU基因序列的转录后修饰。我们提供了直接证据，表明肌苷是酵母异亮氨酰 - tRNA合成酶的正向决定因素。我们还表明，在摆动位置带有鸟苷的酵母tRNAMet会被异亮氨酸氨酰化，同时失去甲硫氨酸接纳能力。由于肌苷和鸟苷共享6 - 酮基和N - 1氢基团，这表明这些氢供体和受体基团是异亮氨酸特异性的决定因素。次要的tRNAIle反密码子假尿苷在tRNA异亮氨酰化中的作用无法直接测试，但可从未修饰转录本活性降低40倍推断得出。鸟苷、肌苷、假尿苷和赖氨酸idine中NHCO结构的存在表明了酵母和大肠杆菌异亮氨酰 - tRNA合成酶对摆动碱基识别的统一模型。与将tRNA识别从甲硫氨酸转换为异亮氨酸的赖氨酸idine不同[村松，T.，西川，K.，根本，F.，串野，Y.，西村，S.，宫泽，T.，&横山，S.（1988年）《自然》336，179 - 181]，假尿苷 - 34不会改变酵母次要tRNAIle的特异性，因为U - 34是酵母甲硫氨酰 - tRNA合成酶（MetRS）的强负向决定因素。因此，Psi - 34（无论是否与Psi - 36结合）的主要作用可能在于异亮氨酸AUA密码子特异性和翻译保真度。
