Wübbeler W, Lossow C, Fittler F, Zachau H G
Eur J Biochem. 1975 Nov 15;59(2):405-13. doi: 10.1111/j.1432-1033.1975.tb02468.x.
When the CCA-halves of tRNAAla1(yeast) and tRNAVal1(Escherichia coli) were incubated with the pG-halves of tRNAVal1 (E.coli) and trnaala1 (yeast), respectively, heterologous complexes were detected. When a 10-fold excess of one half was applied, up to 50% of the other half could be complexed. 5--12% alanine and valine incorporation was observed into the heterolgous combinations in which the pG-halves were derived from tRNAAla1 (yeast) and tRNAVal1 (E.coli), respectively. Although the values are small they appear to be significant considering the results of a number of control experiments. The CCA-half of tRNASer1,2(yeast) and another fragment of this tRNA which extends from the dihydrouridine region to the CCA-terminus were inactive in the aminoacylation assay but they could be converted into a form which accepted serine under standard conditions even in the absence of a complementary fragment. One activation procedure involved the addition of MgCl2 to Mg2+-free fragment solutions, the other consisted in a brief heating-cooling cycle of the fragment solutions at low Mg2+ concentrations. With the two procedures up to 20% or up to 40%, respectively, of the maximal serine incorporation were achieved. At 37 degrees C the active conformation of the fragments persisted only for a few minutes. Analogously, the CCA-halves of tRNAPhe (yeast), tRNAAla1 (yeast), and tRNAVal1 (E.coli)could be activated although here the extent of aminoacylation varied greatly from one experiment to the other. Mischarging of the activated CCA-halves of tRNASer1,2 (yeast) and tRNAPhe (yeast) with phenylalanine and serine, respectively, was not observed. The results obtained with the hererologous fragment combinations and with the CCA-halves alone, which at first sight seem to contradict each other, are discussed with respect to the conformational requirements of synthetase-tRNA recognition.
当分别将酵母tRNAAla1和大肠杆菌tRNAVal1的CCA半分子与大肠杆菌tRNAVal1和酵母tRNAAla1的pG半分子一起温育时，检测到了异源复合物。当加入10倍过量的一种半分子时，高达50%的另一种半分子能够形成复合物。在分别以酵母tRNAAla1和大肠杆菌tRNAVal1的pG半分子形成的异源组合中，观察到有5% - 12%的丙氨酸和缬氨酸掺入。尽管这些值很小，但考虑到一些对照实验的结果，它们似乎具有显著性。酵母tRNASer1,2的CCA半分子以及该tRNA从二氢尿嘧啶区域延伸至CCA末端的另一个片段在氨酰化测定中无活性，但即使在没有互补片段的情况下，它们也能在标准条件下转化为能接受丝氨酸的形式。一种激活方法是向无镁离子的片段溶液中添加氯化镁，另一种方法是在低镁离子浓度下对片段溶液进行短暂的加热 - 冷却循环。通过这两种方法，分别可达到最大丝氨酸掺入量的20%或40%。在37℃时，片段的活性构象仅持续几分钟。类似地，酵母tRNAPhe、酵母tRNAAla1和大肠杆菌tRNAVal1的CCA半分子也能被激活，尽管在此处氨酰化程度在不同实验中差异很大。未观察到被激活的酵母tRNASer1,2和酵母tRNAPhe的CCA半分子分别被苯丙氨酸和丝氨酸错误氨酰化的情况。针对合成酶 - tRNA识别的构象要求，讨论了异源片段组合以及单独的CCA半分子所获得的结果，这些结果乍一看似乎相互矛盾。
