McClain W H, Foss K
Department of Bacteriology, University of Wisconsin, Madison 53706.
J Mol Biol. 1988 Aug 20;202(4):697-709. doi: 10.1016/0022-2836(88)90551-7.
A series of sequence variants of amber suppressor genes of tRNA(Phe) were synthesized in vitro and cloned in Escherichia coli to examine the contributions of individual nucleotides to identity for amino acid acceptance. Three different but complementary types of tRNA variants were constructed. The first involved the substitution of base-pairs on the cloverleaf stem regions of the E. coli tRNA(Phe). The second type of variant involved total gene synthesis based on wild-type tRNA(Phe) sequences found in Bacillus subtilis and in Halobacterium volcanii. In the third type of variant, the identity of E. coli tRNALys was changed to that of tRNA(Phe). The nucleotides which are important for tRNA(Phe) identity in E. coli are located on the corner of the L-shaped tRNA molecule, where the dihydrouridine loop interacts with the T loop, and extend to the interior opening of the anticodon stem and the adjoining variable loop. The nucleotide sequence on the dihydrouridine stem region, which joins the corner and stem regions, was not successfully studied though it may contribute to tRNA(Phe) identity. The fourth nucleotide from the 3' end of tRNA(Phe) has some importance for identity.
体外合成了一系列tRNA（Phe）琥珀抑制基因的序列变体，并将其克隆到大肠杆菌中，以研究单个核苷酸对氨基酸接纳特异性的贡献。构建了三种不同但互补的tRNA变体类型。第一种涉及大肠杆菌tRNA（Phe）三叶草叶茎区域碱基对的替换。第二种变体类型涉及基于枯草芽孢杆菌和火山嗜盐菌中发现的野生型tRNA（Phe）序列进行全基因合成。在第三种变体类型中，将大肠杆菌tRNALys的特异性改变为tRNA（Phe）的特异性。在大肠杆菌中对tRNA（Phe）特异性重要的核苷酸位于L形tRNA分子的转角处，其中二氢尿嘧啶环与T环相互作用，并延伸至反密码子茎的内部开口和相邻的可变环。连接转角和茎区域的二氢尿嘧啶茎区域上的核苷酸序列虽可能对tRNA（Phe）特异性有贡献，但未成功研究。tRNA（Phe）3'端的第四个核苷酸对特异性有一定重要性。
