Mechanistic Basis for Amino Acid Discrimination by the Translational Machinery

翻译机器区分氨基酸的机制基础

• 批准号: 8005733
• 负责人: VIRGINIA W CORNISH
• 金额: $ 37.32万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8005733
• 关键词: Amino Acid Sequence; Amino Acids; Amino Acyl Transfer RNA; Antibiotics; Anticodon; Arts; Belief; Binding Sites; Biochemistry; Biological; Biological Metamorphosis; Cells; Charge; Chemical Structure; Codon Nucleotides; DNA Sequence Rearrangement; Discrimination; Electrostatics; Engineering; Eukaryota; Event; Goals; Image; In Vitro; Investigation; Laboratories; Length; Life; Link; Messenger RNA; Methods; Modeling; Molecular; Movement; Mutagenesis; Pathway interactions; Peptide Elongation Factor Tu; Peptides; Peptidyltransferase; Play; Prokaryotic Cells; Protein Biosynthesis; Proteins; Reaction; Reporting; Research; Ribosomes; Role; Series; Side; Site; Specificity; Structure; Substrate Specificity; System; Testing; Therapeutic; Transfer RNA; Translations; Vertebral column; analog; base; blind; conformational conversion; design; electronic structure; fluorophore; human disease; polypeptide; protein misfolding; public health relevance; research study; stereochemistry

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.

描述（由申请人提供）：核糖体如何在单个催化器中使用超过20种化学上不同的氨基酰基-TRNA底物来合成蛋白质，仍然是一个基本的生物学问题。原始适配器假设指出，AA-TRNA底物特异性完全来自TRNA反密码子与mRNA密码子的相互作用。然而，最近的研究表明，远远超出反dant的tRNA适配器的特征在调节AA-tRNA选择方面也起着关键作用。由于其假定的沉默作用，AA-TRNA对底物选择的氨基酸成分的贡献实际上尚未探索。尽管如此，转化机械对氨基酸视而不见的假设与使用MisacyAcypated TRNA的不良氨基酸数量越来越多。即使对天然氨基酸的化学结构的微妙扰动也可以极大地阻止蛋白质合成。因此，我们假设与适配器假设相反，核糖体不仅针对tRNA适配器，而且对于共同附着在tRNA上的氨基酸的结构和静电剂也是如此。在这里，我们建议通过确定翻译周期中的哪个步骤排除主链类似物，带电的侧链类似物以及WT氨基酸底物的大型侧链类似物来检验这一假设。这些研究的结果应广泛影响努力，以设计非自然的AA-TRNA和翻译机制，以扩大可以使用MisacyAcy tRNA进行合并的类似物的范围，以及我们对AA-TRNA本身在调节构型转变中的作用的基本理解，从而使蛋白质合成。 公共卫生相关性：这项研究的长期目标是（1）为将生物物理探针在细胞中合成时直接掺入蛋白质和（2）以通过核糖体对蛋白质同步机理的基本了解时，直接将生物物理探针掺入蛋白质中。正如用于研究生物分子体外生物分子的生物物理方法一样，对我们对生物分子结构，功能以及为人类疾病开发有效疗法的能力的基本了解，通过直接纳入活细胞中的蛋白质网络的能力，通过不自然的氨基酸荧光酚池和其他生物学探针的潜力和其他生物学的理解能力，使我们的理解能够促进生物含量和其他生物疾病的能力。由于核糖体蛋白质合成是一种主要的细胞途径，因此对该途径的基本理解显着影响了我们根据原核生物和真核生物中这种途径扰动这种途径的能力来开发抗生素和其他类别的治疗剂的能力。