Soma A, Kumagai R, Nishikawa K, Himeno H
Department of Biology, Faculty of Science, Hirosaki University, Japan.
J Mol Biol. 1996 Nov 15;263(5):707-14. doi: 10.1006/jmbi.1996.0610.
The recognition of tRNALeu, one of the class II tRNAs having a long variable arm, by leucyl-tRNA synthetase in Saccharomyces cerevisiae was studied using the T7 transcription system. Exchanging the anticodon arm of tRNALeu but not the D- or T psi C-arm to that of tRNASer seriously affected the leucine accepting activity. Two nucleotides in the anticodon loop, A35 and G37, were found to be important for leucylation. It was also found that the discriminator base, A73, is required for leucylation, and G73 of tRNASer functions as a negative identity determinant for leucyl-tRNA synthetase. Introducing a set of three base substitutions at positions 35, 37 and 73 was sufficient to convert tRNASer into an efficient leucine acceptor. These results indicate that the identity elements of tRNALeu lie at the second position of the anticodon and the 3' adjacent to the anticodon as well as the discriminator position. Such a sequence specific recognition manner is significantly different from that of Escherichia coli, in which not the anticodon but the tertiary structural elements play a key role in discriminating from other class II tRNAs. The leucine system is the first example which shows that the requirement of the anticodon sequence is variable among species.
利用T7转录系统，对酿酒酵母中亮氨酰 - tRNA合成酶识别tRNALeu（具有长可变臂的II类tRNA之一）的情况进行了研究。将tRNALeu的反密码子臂而非D臂或TψC臂替换为tRNASer的相应臂，会严重影响亮氨酸接纳活性。发现反密码子环中的两个核苷酸A35和G37对亮氨酰化很重要。还发现鉴别碱基A73是亮氨酰化所必需的，而tRNASer的G73作为亮氨酰 - tRNA合成酶的负性识别决定因素。在第35、37和73位引入一组三个碱基的替换足以将tRNASer转变为高效的亮氨酸接纳体。这些结果表明，tRNALeu的识别元件位于反密码子的第二位、反密码子相邻的3'端以及鉴别位置。这种序列特异性识别方式与大肠杆菌的显著不同，在大肠杆菌中，不是反密码子而是三级结构元件在区分其他II类tRNA中起关键作用。亮氨酸系统是第一个表明反密码子序列的要求在不同物种间存在差异的例子。
