Anderson J Christopher, Schultz Peter G
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Biochemistry. 2003 Aug 19;42(32):9598-608. doi: 10.1021/bi034550w.
Recently, it has been shown that an amber suppressor tRNA/aminoacyl-tRNA synthetase pair derived from the tyrosyl-tRNA synthetase of Methanococcus jannaschii can be used to genetically encode unnatural amino acids in response to the amber nonsense codon, TAG. However, we have been unable to modify this pair to decode either the opal nonsense codon, TGA, or the four-base codon, AGGA, limiting us to a 21 amino acid code. To overcome this limitation, we have adapted a leucyl-tRNA synthetase from Methanobacterium thermoautotrophicum and leucyl tRNA derived from Halobacterium sp. NRC-1 as an orthogonal tRNA-synthetase pair in Escherichia coli to decode amber (TAG), opal (TGA), and four-base (AGGA) codons. To improve the efficiency and selectivity of the suppressor tRNA, extensive mutagenesis was performed on the anticodon loop and acceptor stem. The two most significant criteria required for an efficient amber orthogonal suppressor tRNA are a CU(X)XXXAA anticodon loop and the lack of noncanonical or mismatched base pairs in the stem regions. These changes afford only weak suppression of TGA and AGGA. However, this information together with an analysis of sequence similarity of multiple native archaeal tRNA sequences led to efficient, orthogonal suppressors of opal codons and the four-base codon, AGGA. Ultimately, it should be possible to use these additional orthogonal pairs to genetically incorporate multiple unnatural amino acids into proteins.
最近的研究表明，源自詹氏甲烷球菌酪氨酰 - tRNA合成酶的琥珀色抑制tRNA/氨酰 - tRNA合成酶对可用于遗传编码非天然氨基酸，以响应琥珀色无义密码子TAG。然而，我们无法对该对进行改造以解码乳白无义密码子TGA或四碱基密码子AGGA，这将我们限制在了21种氨基酸编码。为克服这一限制，我们采用了嗜热自养甲烷杆菌的亮氨酰 - tRNA合成酶和源自嗜盐菌属NRC - 1的亮氨酰tRNA，作为大肠杆菌中的一对正交tRNA - 合成酶，以解码琥珀色（TAG）、乳白（TGA）和四碱基（AGGA）密码子。为提高抑制性tRNA的效率和选择性，我们对反密码子环和受体茎进行了广泛的诱变。高效琥珀色正交抑制性tRNA所需的两个最重要标准是CU(X)XXXAA反密码子环以及茎区域中不存在非规范或错配碱基对。这些变化对TGA和AGGA仅产生微弱的抑制作用。然而，这些信息与对多个天然古菌tRNA序列的序列相似性分析一起，产生了乳白密码子和四碱基密码子AGGA的高效正交抑制子。最终，应该有可能使用这些额外的正交对将多种非天然氨基酸遗传掺入蛋白质中。
