A Kinetic Safety Gate Controlling the Delivery of Unnatural Amino Acids to the Ribosome
A Kinetic Safety Gate Controlling the Delivery of Unnatural Amino Acids to the Ribosome
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DOI：
10.1021/ja407511q
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发表时间：
2013-11-13
影响因子：
15
通讯作者：
Rodnina, Marina V.
中科院分区：
文献类型：
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作者：
Mittelstaet, Joerg;Konevega, Andrey L.;Rodnina, Marina V.
提高非天然氨基酸掺入的产率是生产具有独特化学性质的新型设计蛋白质的一个重要挑战。在这里，我们研究的机制，限制纳入的荧光非天然氨基酸NH 2-Bodipy 576/589-赖氨酸（BOP-Lys）的模型蛋白质。虽然BOP-Lys-tRNA（Lys）向核糖体的递送受到其与延伸因子Tu（EF-Tu）的不良结合的限制，但是在BOP-Lys-tRNA从EF-Tu释放到核糖体中的步骤中，掺入肽的产率被另外控制。非天然氨基酸似乎破坏了平衡tRNA与EF-Tu-GTP结合的强度与tRNA从核糖体上的EF-Tu-GDP解离的速度的相互作用，这确保了标准氨基酸的均匀掺入。规避这种潜在的质量控制检查点，特别是防止非天然氨基酸掺入蛋白质，可以提供一种新的策略，以增加非天然聚合物的产量。
Improving the yield of unnatural amino acid incorporation is an important challenge in producing novel designer proteins with unique chemical properties. Here we examine the mechanisms that restrict the incorporation of the fluorescent unnatural amino acid epsilon NH2-Bodipy576/589-lysine (BOP-Lys) into a model protein. While the delivery of BOP-Lys-tRNA(Lys) to the ribosome is limited by its poor binding to elongation factor Tu (EF-Tu), the yield of incorporation into peptide is additionally controlled at the step of BOP-Lys-tRNA release from EF-Tu into the ribosome. The unnatural amino acid appears to disrupt the interactions that balance the strength of tRNA binding to EF-Tu-GTP with the velocity of tRNA dissociation from EF-Tu-GDP on the ribosome, which ensure uniform incorporation of standard amino acids. Circumventing this potential quality control checkpoint that specifically prevents incorporation of unnatural amino acids into proteins may provide a new strategy to increase yields of unnatural polymers.