Logical engineering of D-arm and T-stem of tRNA that enhances d-amino acid incorporation.
Logical engineering of D-arm and T-stem of tRNA that enhances d-amino acid incorporation.
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DOI：
10.1093/nar/gkx1129
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发表时间：
2017-12-15
影响因子：
14.9
通讯作者：
Suga H
中科院分区：
文献类型：
--
作者：
Katoh T;Iwane Y;Suga H
细菌翻译因子EF-P通过加速Pro-Pro的形成来消除聚脯氨酸序列引起的核糖体停滞。EF-P识别在tRNAPro异源受体中发现的特异性D臂基序，9-nt D环由稳定的D茎序列封闭，用于Pro选择性肽基转移加速。还已知氨酰-tRNA上的T-茎序列调节与EF-Tu相互作用的强度，从而增强非蛋白质氨基酸如一些N-甲基氨基酸的掺入。基于上述认识，我们对tRNA的D-臂和T-茎序列进行了逻辑改造，以研究一系列用于改善d-氨基酸和α，α-二取代氨基酸连续掺入的tRNA。我们设计了tRNAPro1和tRNAGluE2的嵌合体，称为tRNAPro1E2，其中tRNAGluE2的T-茎被工程化到tRNAPro1中。EF-P与预装载有d-Phe、d-Ser、d-Ala和/或d-Cys的tRNAPro1E2NNN的组合与使用正交tRNA的先前方法相比，不仅显著提高了线性肽的表达水平，而且显著提高了由四个连续d-氨基酸组成的硫醚-大环肽的表达水平。
A bacterial translation factor EF-P alleviates ribosomal stalling caused by polyproline sequence by accelerating Pro-Pro formation. EF-P recognizes a specific D-arm motif found in tRNAPro isoacceptors, 9-nt D-loop closed by a stable D-stem sequence, for Pro-selective peptidyl-transfer acceleration. It is also known that the T-stem sequence on aminoacyl-tRNAs modulates strength of the interaction with EF-Tu, giving enhanced incorporation of non-proteinogenic amino acids such as some N-methyl amino acids. Based on the above knowledge, we logically engineered tRNA’s D-arm and T-stem sequences to investigate a series of tRNAs for the improvement of consecutive incorporation of d-amino acids and an α, α-disubstituted amino acid. We have devised a chimera of tRNAPro1 and tRNAGluE2, referred to as tRNAPro1E2, in which T-stem of tRNAGluE2 was engineered into tRNAPro1. The combination of EF-P with tRNAPro1E2NNN pre-charged with d-Phe, d-Ser, d-Ala, and/or d-Cys has drastically enhanced expression level of not only linear peptides but also a thioether-macrocyclic peptide consisting of the four consecutive d-amino acids over the previous method using orthogonal tRNAs.
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影响因子：
15
作者：
Doerfel, Lili K.;Wohlgemuth, Ingo;Rodnina, Marina V.
通讯作者：
Rodnina, Marina V.
DOI：
10.1073/pnas.1102128108
发表时间：
2011-03-29
影响因子：
11.1
作者：
Schrader, Jared M.;Chapman, Stephen J.;Uhlenbeck, Olke C.
通讯作者：
Uhlenbeck, Olke C.
影响因子：
4.5
作者：
Goto, Yuki;Murakami, Hiroshi;Suga, Hiroaki
通讯作者：
Suga, Hiroaki
影响因子：
2.9
作者：
Dedkova, Larisa M.;Fahmi, Nour Eddine;Hecht, Sidney M.
通讯作者：
Hecht, Sidney M.
影响因子：
--
作者：
Ohta, Atsushi;Murakami, Hiroshi;Suga, Hiroaki
通讯作者：
Suga, Hiroaki