Mechanistic Basis for Amino Acid Discrimination by the Translational Machinery

描述(由申请人提供)：核糖体如何在单一催化装置中使用20多种不同的化学上不同的氨酰-tRNA底物来合成蛋白质仍然是一个基本的生物学问题。最初的接头假说认为，AA-tRNA底物的特异性完全来自tRNA反密码子与mRNA密码子的相互作用。然而，最近的研究表明，tRNA接头的功能远远超出反密码子，在调节AA-tRNA选择方面也发挥着关键作用；由于它被认为是沉默的角色，AA-tRNA的氨基酸成分对底物选择的贡献几乎还没有被探索。尽管如此，翻译机制对氨基酸视而不见的假设与越来越多的非自然氨基酸是不一致的，这些非自然氨基酸使用错酰化的tRNA结合得很差；即使是对天然氨基酸化学结构的细微扰动也会显著阻止蛋白质的合成。因此，我们假设，与接头假说相反，核糖体不仅对tRNA接头具有微妙的特异性，而且对与tRNA共价结合的氨基酸的结构和静电学也是特异的。在这里，我们建议通过确定翻译周期中的哪个步骤(S)排除wt氨基酸底物的主干类似物、带电侧链类似物和大侧链类似物来检验这一假设。这些研究的结果应该会广泛地影响到设计非自然的AA-tRNA和翻译机制的努力，以扩大可以使用错酰化的tRNA结合的类似物的范围，以及我们对AA-tRNA本身在调节蛋白质合成基础的构象转变中所起作用的基本理解。 与公共健康相关：这项研究的长期目标是(1)设计将生物物理探针直接结合到蛋白质中的翻译机制，因为它们是在细胞中合成的；(2)对核糖体合成蛋白质的机制有一个基本的了解。正如体外研究生物分子的生物物理方法显著影响了我们对生物分子结构和功能的基本理解以及开发有效治疗人类疾病的药物的能力一样，通过直接掺入非天然氨基酸荧光团和其他生物物理探针来成像活细胞中的蛋白质网络的能力可能对我们理解生物途径和人类疾病的机制做出广泛而重大的贡献。由于核糖体的蛋白质合成是一条主要的细胞途径，因此对这一途径的基本了解将显著影响我们开发抗生素和其他类型的治疗药物的能力，这些药物基于它们在原核生物和真核生物中干扰这一途径的能力。

DESCRIPTION (provided by applicant): How the ribosome can use over twenty chemically distinct aminoacyl-tRNA substrates within a single catalytic apparatus to synthesize proteins remains a fundamental biological question. The original adaptor hypothesis states that aa- tRNA substrate specificity comes entirely from the interaction of the tRNA anticodon with the mRNA codon. Recent studies, however, have revealed that features of the tRNA adaptor well beyond the anticodon also play critical roles in regulating aa-tRNA selection; due to its assumed silent role, the contribution of the amino acid component of the aa-tRNA to substrate selection remains virtually unexplored. Despite this, the hypothesis that the translational machinery is blind to the amino acid is at odds with the growing number of unnatural amino acids that are poorly incorporated using misacylated tRNAs; even subtle perturbations to the chemical structures of the natural amino acids can dramatically arrest protein synthesis. Thus, we hypothesize that, contrary to the adaptor hypothesis, the ribosome is exquisitely specific not only for the tRNA adaptor, but also for the structure and electrostatics of the amino acid covalently attached to the tRNA. Here, we propose to test this hypothesis by determining which step(s) in the translation cycle exclude backbone analogs, charged side-chain analogs, and large side-chain analogs of the wt amino acid substrates. The results of these studies should broadly impact efforts to engineer the unnatural aa-tRNA and the translational machinery to expand the range of analogs that can be incorporated using misacylated tRNAs and our fundamental understanding of the role of the aa-tRNA itself in regulating the conformational transitions that underlie protein synthesis. PUBLIC HEALTH RELEVANCE: The long term goals of this research are (1) to engineer the translational machinery for the incorporation of biophysical probes into proteins directly as they are being synthesized in the cell and (2) to gain a fundamental understanding of the mechanism of protein synthesis by the ribosome. Just as biophysical methods for studying biomolecules in vitro have significantly impacted our fundamental understanding of biomolecule structure and function and ability to develop effective therapeutics for human disease, the ability to image protein networks in living cells by direct incorporation of unnatural amino acid fluorophores and other biophysical probes has the potential to make broad, significant contributions to our understanding of the mechanism of biological pathways and human disease. Because protein synthesis by the ribosome is a major cellular pathway, fundamental understanding of this pathway significantly impacts our ability to develop antibiotics and other classes of therapeutics based on their ability to perturb this pathway both in prokaryotes and eukaryotes.