Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA.
Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA.
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DOI：
10.1093/nar/gkq521
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发表时间：
2010-10
影响因子：
14.9
通讯作者：
Ellington AD
中科院分区：
文献类型：
--
作者：
Hughes RA;Ellington AD
虽然已经有许多氨基酰基tRNA合成酶(AARs)：tRNA对被设计来改变或扩展遗传密码，但只有詹氏酪氨酸甲酯tRNA合成酶和tRNA在细菌中被广泛使用，限制了任何时候可以用来扩展遗传密码的非天然氨基酸的类型和数量。为了扩大可用于细菌遗传密码工程的AARS/tRNA对的数量和类型，我们从酿酒酵母中开发了一种正交色氨基tRNA合成酶和tRNA对。在开发琥珀抑制tRNA的过程中，我们发现大肠杆菌赖氨酰tRNA合成酶负责错酰化最初的琥珀抑制版本的酵母色氨酸tRNA。人们发现，修改反密码子茎的G：C含量，从而降低该茎的结构灵活性，消除了大肠杆菌赖氨酰tRNA合成酶的错酰化，并导致了一对功能性的、正交的抑制子对的发展，这应该被证明是有用的，大量的，非天然的氨基酸纳入遗传密码。我们的结果为tRNA灵活性在分子识别和tRNA特异性的工程和进化中的作用提供了洞察力。
While a number of aminoacyl tRNA synthetase (aaRS):tRNA pairs have been engineered to alter or expand the genetic code, only the Methanococcus jannaschii tyrosyl tRNA synthetase and tRNA have been used extensively in bacteria, limiting the types and numbers of unnatural amino acids that can be utilized at any one time to expand the genetic code. In order to expand the number and type of aaRS/tRNA pairs available for engineering bacterial genetic codes, we have developed an orthogonal tryptophanyl tRNA synthetase and tRNA pair, derived from Saccharomyces cerevisiae. In the process of developing an amber suppressor tRNA, we discovered that the Escherichia coli lysyl tRNA synthetase was responsible for misacylating the initial amber suppressor version of the yeast tryptophanyl tRNA. It was discovered that modification of the G:C content of the anticodon stem and therefore reducing the structural flexibility of this stem eliminated misacylation by the E. coli lysyl tRNA synthetase, and led to the development of a functional, orthogonal suppressor pair that should prove useful for the incorporation of bulky, unnatural amino acids into the genetic code. Our results provide insight into the role of tRNA flexibility in molecular recognition and the engineering and evolution of tRNA specificity.
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