Modified Nucleoside Structure-Function Relations: tRNA

修饰核苷结构-功能关系：tRNA

• 批准号: 6641850
• 负责人: PAUL F AGRIS
• 金额: $ 6万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-01-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6641850
• 关键词: aminoacid tRNA ligase; calorimetry; chemical structure function; chemical synthesis; circular dichroism; fluorescence spectrometry; genetic translation; intermolecular interaction; messenger RNA; model design /development; molecular shape; molecular site; nuclear magnetic resonance spectroscopy; nucleic acid sequence; nucleoside analog; nucleosides; physical model; protein biosynthesis; protein sequence; ribonucleoproteins; ribosomes; structural biology; thermodynamics; thermostability; transfer RNA

RNA's modified nucleosides are essential to protein synthesis. However, too little is known of the biochemical and structural contributions of the 100 natural, post-transcriptional modifications to relate structure to function. Fundamental understandings of modified nucleoside functions have shown promise in identifying novel therapeutic targets in infectious disease, and in optimizing ribozyme, antisense and aptamer activities. Thus, this project's long-term objective is to understand how the biochemical and structural contributions of modified nucleosides impact protein recognition of RNA and its function in translation. tRNA, composed of physically and functionally separable domains that are easily investigated for modification-dependent structure/function relationships, is an excellent model for this study. We hypothesize that anticodon modified nucleosides provide a common architecture for stably recognizing mRNA coding triplets on the ribosome, while at the same time providing hydrophobic, hydrophilic and/or electrostatic properties as identity elements for aminoacyl-tRNA synthetase (aaRS) recognition. Using methods developed almost exclusively by us for the automated chemical synthesis of RNA with site- selectively placed modified and stable isotope labeled nucleosides, we have found three functionally important modified nucleosides that modulate anticodon loop architecture. To determine the structure/function relationships of other anticodon domain modifications, we will: 1) Identify and characterize the modified nucleoside-dependent ribosomal binding of tRNA anticodon domains that do not bind in their unmodified sequences; 2) Determine and characterize the properties of modified anticodon domains that are selectively recognized by peptides as mimics of aaRS; and 3) Characterize the structural properties contributed by modified nucleosides to the ribosomal binding and aaRS recognition of anticodons. Our approach will utilize a unique combination of chemical synthesis, biochemical assays of function and thermal denaturation, calorimetry, fluorescence, CD and NMR spectroscopy for determining stability and structure to distinguish those modifications required for ribosome binding and aminoacylation and their critical physiochemical contributions.

RNA 的修饰核苷对于蛋白质合成至关重要。 然而，对于将结构与功能联系起来的 100 种天然转录后修饰的生化和结构贡献知之甚少。 对修饰核苷功能的基本了解在确定传染病的新治疗靶点以及优化核酶、反义和适体活性方面显示出了希望。 因此，该项目的长期目标是了解修饰核苷的生化和结构贡献如何影响 RNA 的蛋白质识别及其翻译功能。 tRNA 由物理上和功能上可分离的结构域组成，很容易研究修饰依赖性结构/功能关系，是本研究的绝佳模型。 我们假设反密码子修饰的核苷提供了一个共同的结构，用于稳定识别核糖体上编码三联体的 mRNA，同时提供疏水性、亲水性和/或静电特性作为氨酰基-tRNA 合成酶 (aaRS) 识别的识别元件。 使用几乎完全由我们开发的用于自动化学合成RNA的方法，该方法具有位点选择性放置的修饰和稳定同位素标记的核苷，我们发现了三种功能上重要的调节反密码子环结构的修饰核苷。 为了确定其他反密码子结构域修饰的结构/功能关系，我们将： 1) 鉴定并表征在其未修饰序列中不结合的 tRNA 反密码子结构域的修饰核苷依赖性核糖体结合； 2) 确定并表征修饰的反密码子结构域的特性，这些结构域被肽选择性识别为 aaRS 的模拟物； 3) 表征修饰核苷对核糖体结合和反密码子 aaRS 识别所贡献的结构特性。 我们的方法将利用化学合成、功能生化测定和热变性、量热法、荧光、CD和NMR光谱的独特组合来确定稳定性和结构，以区分核糖体结合和氨酰化所需的修饰及其关键的理化贡献。