Hohsaka T, Ashizuka Y, Murakami H, Sisido M
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
Nucleic Acids Res. 2001 Sep 1;29(17):3646-51. doi: 10.1093/nar/29.17.3646.
Extension of the genetic code for the introduction of nonnatural amino acids into proteins was examined by using five-base codon-anticodon pairs. A streptavidin mRNA containing a CGGUA codon at the Tyr54 position and a tRNA(UACCG) chemically aminoacylated with a nonnatural amino acid were added to an Escherichia coli in vitro translation system. Western blot analysis indicated that the CGGUA codon is decoded by the aminoacyl-tRNA containing the UACCG anticodon. HPLC analysis of the tryptic fragment of the translation product revealed that the nonnatural amino acid was incorporated corresponding to the CGGUA codon without affecting the reading frame adjacent to the CGGUA codon. Another 15 five-base codons CGGN(1)N(2), where N(1) and N(2) indicate one of four nucleotides, were also successfully decoded by aminoacyl-tRNAs containing the complementary five-base anticodons. These results provide a novel strategy for nonnatural mutagenesis as well as a novel insight into the mechanism of frameshift suppression.
通过使用五碱基密码子 - 反密码子对来研究将非天然氨基酸引入蛋白质中的遗传密码扩展。将在Tyr54位置含有CGGUA密码子的链霉亲和素mRNA和用非天然氨基酸化学氨酰化的tRNA（UACCG）添加到大肠杆菌体外翻译系统中。蛋白质免疫印迹分析表明，CGGUA密码子由含有UACCG反密码子的氨酰 - tRNA解码。对翻译产物的胰蛋白酶片段进行HPLC分析表明，非天然氨基酸对应于CGGUA密码子被掺入，而不影响与CGGUA密码子相邻的阅读框。另外15个五碱基密码子CGGN(1)N(2)（其中N(1)和N(2)表示四种核苷酸之一）也被含有互补五碱基反密码子的氨酰 - tRNA成功解码。这些结果为非天然诱变提供了一种新策略，同时也为移码抑制机制提供了新的见解。
