Synthesis of Peptidyl-tRNA Mimics for Structural Biology Applications.
Synthesis of Peptidyl-tRNA Mimics for Structural Biology Applications.
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DOI：
10.1021/acs.accounts.3c00412
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发表时间：
2023-10-03
影响因子：
18.3
通讯作者：
Micura, Ronald
中科院分区：
文献类型：
--
作者：
Polikanov, Yury S.;Etheve-Quelquejeu, Meïlanie;Micura, Ronald
蛋白质生物合成是所有活细胞中的一个核心过程，由复杂的分子机器——核糖体催化。这个过程被称为翻译，因为 mRNA 中的核苷酸语言被翻译成蛋白质中的氨基酸语言。带有氨基酸的转移 RNA (tRNA) 分子充当接头并在解码中心识别 mRNA 的密码子，同时各个氨基酸在肽基转移酶中心 (PTC) 中组装成肽链。当新生肽从核糖体中出现时，它会穿过一条称为新生肽出口隧道 (NPET) 的长隧道。 PTC 和 NPET 是许多抗生素的靶位点，因此从生物医学角度和制药行业的药物开发而言具有极其重要的意义。研究人员在分子水平上表征核糖体翻译方面取得了很大进展；现在可以获得数量惊人的核糖体不同功能和抑制状态的高分辨率结构。这些结构极大地促进了我们对核糖体如何与其关键底物（即 mRNA、tRNA 和翻译因子）相互作用的理解。相比之下，人们对小分子（尤其是抗生素）如何影响核糖体蛋白质合成的机制知之甚少。这主要涉及小分子-NPET干扰共翻译蛋白质折叠和蛋白质合成调节的结构基础。越来越多的生化证据表明，NPET 在蛋白质合成的调节中发挥着积极作用。该领域急需的进展受到以下事实的阻碍：在制备用于结构研究（即 X 射线晶体学、冷冻电子显微镜和核磁共振波谱）的核糖体复合物过程中，氨酰基或肽基 tRNA 不稳定并被水解。该问题的一个解决方案是应用氨酰基-或肽基-tRNA 的抗水解模拟物。在本篇文章中，我们概述了生成肽基-tRNA 类似物的合成方法。已经开发出模块化方法，将（i）在3'-氨基酰氨基-腺苷树脂上进行RNA和肽固相合成，（ii）天然化学连接和施陶丁格连接，（iii）通过用DNA酶选择性切割天然天然tRNA来定制tRNA，然后通过酶连接重新组装成合成的肽基-RNA片段，以及（iv）酶促加尾和半胱氨酸充电tRNA，以获得与感兴趣的肽部分化学连接的 tRNA 的修饰 CCA 末端。原则上，有了这个工具库，任何所需的稳定连接的肽基-tRNA 模拟物序列都可以实现。为了强调合成缀合物的重要性，我们简要指出了最关键的应用，这些应用为核糖体靶向抗生素的上下文特异性活性、多个连续脯氨酸残基的核糖体依赖性掺入、d-氨基酸的掺入和 tRNA 错误充电的分子机制提供了新的线索。此外，我们讨论了依赖于三唑和方酸酯（而不是酰胺）连接的缀合物的新型稳定带电 tRNA 类似物。这些推动了我们对非核糖体肽合成机制的理解，其中氨酰基-tRNA依赖性酶关键参与初级和次级代谢以及细菌细胞壁合成的各种细胞过程。
Protein biosynthesis is a central process in all living cells that is catalyzed by a complex molecular machine—the ribosome. This process is termed translation because the language of nucleotides in mRNAs is translated into the language of amino acids in proteins. Transfer RNA (tRNA) molecules charged with amino acids serve as adaptors and recognize codons of mRNA in the decoding center while simultaneously the individual amino acids are assembled into a peptide chain in the peptidyl transferase center (PTC). As the nascent peptide emerges from the ribosome, it is threaded through a long tunnel referred to as a nascent peptide exit tunnel (NPET). The PTC and NPET are the sites targeted by many antibiotics and are thus of tremendous importance from a biomedical perspective and for drug development in the pharmaceutical industry. Researchers have achieved much progress in characterizing ribosomal translation at the molecular level; an impressive number of high-resolution structures of different functional and inhibited states of the ribosome are now available. These structures have significantly contributed to our understanding of how the ribosome interacts with its key substrates, namely, mRNA, tRNAs, and translation factors. In contrast, much less is known about the mechanisms of how small molecules, especially antibiotics, affect ribosomal protein synthesis. This mainly concerns the structural basis of small molecule–NPET interference with cotranslational protein folding and the regulation of protein synthesis. Growing biochemical evidence suggests that NPET plays an active role in the regulation of protein synthesis. Much-needed progress in this field is hampered by the fact that during the preparation of ribosome complexes for structural studies (i.e., X-ray crystallography, cryoelectron microscopy, and NMR spectroscopy) the aminoacyl- or peptidyl-tRNAs are unstable and become hydrolyzed. A solution to this problem is the application of hydrolysis-resistant mimics of aminoacyl- or peptidyl-tRNAs. In this Account, we present an overview of synthetic methods for the generation of peptidyl-tRNA analogs. Modular approaches have been developed that combine (i) RNA and peptide solid-phase synthesis on 3′-aminoacylamino-adenosine resins, (ii) native chemical ligations and Staudinger ligations, (iii) tailoring of tRNAs by the selective cleavage of natural native tRNAs with DNAzymes followed by reassembly with enzymatic ligation to synthetic peptidyl-RNA fragments, and (iv) enzymatic tailing and cysteine charging of the tRNA to obtain modified CCA termini of a tRNA that are chemically ligated to the peptide moiety of interest. With this arsenal of tools, in principle, any desired sequence of a stably linked peptidyl-tRNA mimic is accessible. To underline the significance of the synthetic conjugates, we briefly point to the most critical applications that have shed new light on the molecular mechanisms underlying the context-specific activity of ribosome-targeting antibiotics, ribosome-dependent incorporation of multiple consecutive proline residues, the incorporation of d-amino acids, and tRNA mischarging. Furthermore, we discuss new types of stably charged tRNA analogs, relying on triazole- and squarate (instead of amide)-linked conjugates. Those have pushed forward our mechanistic understanding of nonribosomal peptide synthesis, where aminoacyl-tRNA-dependent enzymes are critically involved in various cellular processes in primary and secondary metabolism and in bacterial cell wall synthesis.
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