Import of amber and ochre suppressor tRNAs into mammalian cells:: A general approach to site-specific insertion of amino acid analogues into proteins
Import of amber and ochre suppressor tRNAs into mammalian cells:: A general approach to site-specific insertion of amino acid analogues into proteins
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DOI：
10.1073/pnas.251438898
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发表时间：
2001-12-04
影响因子：
11.1
通讯作者：
Rajbhandary, UL
中科院分区：
文献类型：
--
作者：
Köhrer, C;Xie, L;Rajbhandary, UL
将氨基酸类似物在体内位点特异性插入蛋白质的一般方法是将与所选类似物氨酰化的抑制性tRNA导入细胞。类似物可以插入到mRNA中终止密码子指定的蛋白质中的任何位点。唯一的要求是抑制tRNA不能是任何细胞氨酰-tRNA合成酶的底物。在这里，我们描述的条件进口琥珀和赭石抑制tRNA来源于大肠杆菌启动子tRNA到哺乳动物COS 1细胞，我们目前的证据表明，他们的活动在琥珀（UAG）和赭石（UAA）密码子的特异性抑制。我们发现，一个氨酰化的琥珀抑制tRNA（supF）来源于大肠杆菌。大肠杆菌酪氨酸tRNA可以输入到COS 1细胞中，并作为琥珀密码子的抑制因子，而没有预先氨酰化的相同抑制因子tRNA则不能输入，这表明supF tRNA不是任何哺乳动物氨酰tRNA合成酶的底物。这些结果打开了使用supF tRNA氨酰化的氨基酸类似物作为一种通用的方法，在哺乳动物细胞中的蛋白质的位点特异性插入的氨基酸类似物的可能性。我们讨论了进一步进口琥珀和赭石抑制tRNA的混合物插入两个不同的氨基酸类似物到一个蛋白质和潜在的使用抑制tRNA进口治疗一些人类遗传疾病引起的无义突变的可能性。
A general approach to site-specific insertion of amino acid analogues into proteins in vivo would be the import into cells of a suppressor tRNA aminoacylated with the analogue of choice. The analogue would be inserted at any site in the protein specified by a stop codon in the mRNA. The only requirement is that the suppressor tRNA must not be a substrate for any of the cellular aminoacyl-tRNA synthetases. Here, we describe conditions for the import of amber and ochre suppressor tRNAs derived from Escherichia coli initiator tRNA into mammalian COS1 cells, and we present evidence for their activity in the specific suppression of amber (UAG) and ochre (UAA) codons, respectively. We show that an aminoacylated amber suppressor tRNA (supF) derived from the E. coli tyrosine tRNA can be imported into COS1 cells and acts as a suppressor of amber codons, whereas the same suppressor tRNA imported without prior aminoacylation does not, suggesting that the supF tRNA is not a substrate for any mammalian aminoacyl-tRNA synthetase. These results open the possibility of using the supF tRNA aminoacylated with an amino acid analogue as a general approach for the site-specific insertion of amino acid analogues into proteins in mammalian cells. We discuss the possibility further of importing a mixture of amber and ochre suppressor tRNAs for the insertion of two different amino acid analogues into a protein and the potential use of suppressor tRNA import for treatment of some of the human genetic diseases caused by nonsense mutations.