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超过20种化学上不同的氨基酰trna底物来合成蛋白质仍然是一个基本的生物学问题。最初的适配器假说认为，aa- tRNA底物特异性完全来自于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接头的特征远不止反密码子，在调节aa-tRNA选择中也起着关键作用；由于假设其沉默作用，aa-tRNA的氨基酸成分对底物选择的贡献实际上尚未被探索。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天然氨基酸不一致，这些非天然氨基酸被misacylated trna不良地结合；即使是对天然氨基酸的化学结构的微小扰动也能极大地阻止蛋白质的合成。因此，我们假设，与接头假说相反，核糖体不仅对tRNA接头，而且对与tRNA共价连接的氨基酸的结构和静电特性都具有精确的特异性。在这里，我们建议通过确定翻译周期中哪些步骤排除主干类似物、带电侧链类似物和wt氨基酸底物的大侧链类似物来验证这一假设。这些研究的结果将广泛影响非自然的aa-tRNA和翻译机制，以扩大可以使用misacylated 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上的构象转变中的作用的基本理解。

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.