Modified Nucleoside Structure-Function Relations: tRNA

RNA的修饰核苷对蛋白质合成至关重要。 然而，对100种天然转录后修饰的生物化学和结构贡献知之甚少，无法将结构与功能联系起来。 对修饰的核苷功能的基本理解在识别感染性疾病的新治疗靶点以及优化核酶、反义核酸和适体活性方面显示出希望。 因此，该项目的长期目标是了解修饰核苷的生物化学和结构贡献如何影响RNA的蛋白质识别及其在翻译中的功能。 tRNA是一个很好的模型，它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的结构/功能关系。 我们假设，反密码子修饰的核苷提供了一个共同的架构，用于稳定地识别核糖体上的mRNA编码三联体，而在同一时间提供疏水性，亲水性和/或静电特性作为氨酰-tRNA合成酶（阿尔斯）识别的身份元素。 使用几乎完全由我们开发的用于自动化化学合成具有位点选择性放置的修饰的和稳定的同位素标记的核苷的RNA的方法，我们发现了三种调节反密码子环结构的功能上重要的修饰的核苷。 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）鉴定和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的核苷依赖性核糖体结合; 2）确定和表征被肽选择性识别为阿尔斯模拟物的修饰的反密码子结构域的性质;和3）表征修饰的核苷对核糖体结合和反密码子的阿尔斯识别所贡献的结构性质。 我们的方法将利用化学合成，功能和热变性，量热法，荧光，CD和NMR光谱的生化测定的独特组合，用于确定稳定性和结构，以区分核糖体结合和氨酰化所需的那些修饰及其关键的理化贡献。

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.