Modified Nucleoside Structure-Function Relations: tRNA

RNA的修饰核苷对蛋白质合成是必不可少的。然而，人们对100种天然的转录后修饰的生化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确定感染性疾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的蛋白质识别及其在翻译中的功能。TRNA由物理和功能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，是这项研究的一个很好的模型。我们假设反密码子修饰的核苷为稳定识别核糖体上编码三联体的mRNA提供了一个共同的体系结构，同时提供了疏水性、亲水性和/或静电特性作为识别氨基酰基-tRNA合成酶(AARS)的识别元件。利用我们开发的几乎完全由我们开发的自动化学合成RNA的方法，并选择性地定位修饰和稳定的同位素标记核苷，我们发现了三种具有重要功能的修饰核苷，它们调节反密码子环结构。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)鉴定和表征未在其未修饰序列中结合的tRNA反密码子结构域的修饰的依赖于核苷的核糖体结合；2)确定和表征被多肽选择性识别为AARs模拟物的修饰的反密码子结构域的性质；以及3)表征修饰的核苷对反密码子的核糖体结合和AARS识别的结构特性。我们的方法将利用化学合成、功能和热变性的生化分析、量热、荧光、CD和核磁共振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基酰化所需的修饰及其关键的物理化学贡献。

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.