BIOCHEMICAL AND PHYSICAL CHARACTERIZATION OF AN UNMODIFIED YEAST PHENYLALANINE TRANSFER-RNA TRANSCRIBED INVITRO
BIOCHEMICAL AND PHYSICAL CHARACTERIZATION OF AN UNMODIFIED YEAST PHENYLALANINE TRANSFER-RNA TRANSCRIBED INVITRO
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DOI：
10.1073/pnas.85.4.1033
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发表时间：
1988-02-01
影响因子：
11.1
通讯作者：
UHLENBECK, OC
中科院分区：
文献类型：
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作者：
SAMPSON, JR;UHLENBECK, OC
用六种合成的DNA寡聚物构建重组质粒，使得对应于酵母tRNAPhe的DNA序列侧接T7启动子和BstNI限制性位点。用T7 RNA聚合酶对BstNI消化的质粒进行溢流转录，得到具有正确5“和3”末端的预期序列的未修饰的tRNA。tRNAPhe转录物可以被酵母苯丙氨酰-tRNA合成酶特异性地氨基丙烯酸化，并且具有仅比天然酵母tRNAPhe高4倍的Km。Km与Mg ~（2+）浓度无关，而Vmax与Mg ~（2+）浓度密切相关。在不同的Mg 2+浓度的本地和未改性的tRNAPhe的熔化曲线的比较表明，未改性的tRNAPhe具有不太稳定的三级结构。使用一个额外的DNA寡聚体，构建了在tRNA基因的第20位具有鸟苷至胸苷变化的突变质粒。观察到突变体tRNAPhe转录物的氨酰化的Vmax/Km降低了14倍。
A recombinant plasmid was constructed with six synthetic DNA oligomers such that the DNA sequence corresponding to yeast tRNAPhe is flanked by a T7 promoter and a BstNI restriction site. Runoff transcription of the BstNI-digested plasmid with T7 RNA polymerase gives an unmodified tRNA of the expected sequence having correct 5'' and 3'' termini. The tRNAPhe transcript can be specifically aminoacrylated by yeast phenylalanyl-tRNA synthetase and has a Km only 4-fold higher than that of the native yeast tRNAPhe. The Km is independent of Mg2+ concentration, whereas the Vmax is very dependent on Mg2+ concentration. Comparison of the melting profiles of the native and the unmodified tRNAPhe at different Mg2+ concentrations suggests that the unmodified tRNAPhe has a less stable tertiary structure. Using one additional DNA oligomer, a mutant plasmid was constructed having a guanosine to thymidine change at position 20 in the tRNA gene. A decrease in Vmax/Km by a factor of 14 for aminoacylation of the mutant tRNAPhe transcript is observed.