Transfer RNA Misidentification Scrambles Sense Codon Recoding
Transfer RNA Misidentification Scrambles Sense Codon Recoding
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DOI：
10.1002/cbic.201300444
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发表时间：
2013-10-11
期刊：
影响因子：
3.2
通讯作者：
Soell, Dieter
中科院分区：
文献类型：
--
作者：
Krishnakumar, Radha;Prat, Laure;Soell, Dieter
正义密码子编码是用两种以上不同的非规范氨基酸进行遗传密码扩展的基础。它需要一个未使用的(或很少使用的)密码子，以及与互补反密码子正交的tRNA合成酶：tRNA对。羊支原体基因组只包含6个CGG精氨酸密码子，没有专门的tRNA(Arg)。我们想通过给微囊藻提供吡咯酰-tRNA合成酶，一个带有CCG反密码子的合成tRNA(tRNA(CCG)(Pyl))和吡咯赖氨酸生物合成的基因，将这个密码子重新分配给吡咯赖氨酸。在这里，我们证明了tRNA(CCG)(PYL)可以有效地被内源性精氨酰-tRNA合成酶识别，推测是在反密码子上。质谱仪显示，在tRNA(CCG)(Pyl)存在下，CGG密码子被翻译为精氨酸。这一结果并不出人意料，因为大多数tRNA合成酶使用反密码子作为识别元件。这些数据表明，内源性氨基酰-tRNA合成酶对tRNA的错误识别需要克服，以进行正义密码子的重新编码。
Sense codon recoding is the basis for genetic code expansion with more than two different noncanonical amino acids. It requires an unused (or rarely used) codon, and an orthogonal tRNA synthetase: tRNA pair with the complementary anticodon. The Mycoplasma capricolum genome contains just six CGG arginine codons, without a dedicated tRNA(Arg). We wanted to reassign this codon to pyrrolysine by providing M. capricolum with pyrrolysyl-tRNA synthetase, a synthetic tRNA with a CCG anticodon (tRNA(CCG)(Pyl)), and the genes for pyrrolysine biosynthesis. Here we show that tRNA(CCG)(Pyl) is efficiently recognized by the endogenous arginyl-tRNA synthetase, presumably at the anticodon. Mass spectrometry revealed that in the presence of tRNA(CCG)(Pyl), CGG codons are translated as arginine. This result is not unexpected as most tRNA synthetases use the anticodon as a recognition element. The data suggest that tRNA misidentification by endogenous aminoacyl-tRNA synthetases needs to be overcome for sense codon recoding.