Asahara H, Himeno H, Tamura K, Hasegawa T, Watanabe K, Shimizu M
Institute of Space and Astronautical Science, Kanagawa, Japan.
J Mol Biol. 1993 May 20;231(2):219-29. doi: 10.1006/jmbi.1993.1277.
In order to study how Escherichia coli leucyl-tRNA synthetase recognizes tRNA(Leu) and discriminates it from the other two class II tRNAs, tRNA(Ser) and tRNA(Tyr), various mutations were introduced into class II tRNA transcripts. The discriminator base A73, but not the anticodon sequence, was found to serve as a critical recognition element of tRNA(Leu). A base substitution at the invariant nucleotide A14, but not at any of the other nucleotides characteristic of the E. coli tRNA(Leu) isoacceptors among the three class II tRNAs, caused significantly damaged aminoacylation with leucine. A two base-pair deletion in the long variable arm also resulted in no significant decrease of activity. Transplanting the three tertiary elements characteristic of E. coli tRNA(Leu) (i.e. the location of the G18G19 sequence in the D-loop, the A15 U48 base-pair and the stem pairing pattern of the long variable arm) besides the discriminator base change introduced the leucine charging activity in terms of Vmax/Km, up to 0.1 of that for the normal sequence of tRNA(Leu) into both tRNA(Ser) and tRNA(Tyr). These results indicate that A73 and A14 (or its vicinity) are involved in recognition by leucyl-tRNA synthetase, and that several tertiary elements play a significant role in the discrimination of tRNA(Leu) from the other two class II tRNAs.
为了研究大肠杆菌亮氨酰 - tRNA合成酶如何识别tRNA(Leu)并将其与另外两种II类tRNA，即tRNA(Ser)和tRNA(Tyr)区分开来，研究人员在II类tRNA转录本中引入了各种突变。结果发现，鉴别碱基A73而非反密码子序列是tRNA(Leu)的关键识别元件。在三种II类tRNA中，大肠杆菌tRNA(Leu)同工受体特有的不变核苷酸A14发生碱基替换会导致亮氨酸氨酰化显著受损，而其他核苷酸发生替换则不会。长可变臂中的两个碱基对缺失也不会导致活性显著降低。除了鉴别碱基变化外，移植大肠杆菌tRNA(Leu)特有的三个三级元件（即D环中G18G19序列的位置、A15 U48碱基对以及长可变臂的茎配对模式），可使tRNA(Ser)和tRNA(Tyr)的亮氨酸负载活性（以Vmax/Km衡量）提高至正常tRNA(Leu)序列的0.1。这些结果表明，A73和A14（或其附近区域）参与亮氨酰 - tRNA合成酶的识别，并且几个三级元件在tRNA(Leu)与其他两种II类tRNA 的区分中起重要作用。
