MODIFIED NUCLEOSIDE STRUCTURE FUNCTION RELATIONS--TRNA

修饰的核苷赋予核酸独特的位点特异性 酸，但确定其结构功能的重要性 关系被证明是困难的。 据推测， 核苷与它们改变RNA化学有关， 结构，实现这一目标的潜力是多种多样的， 修饰的化学和结构。 的理解 独特的化学，结构和动力学，80多个自然 发生的修饰核苷有助于核酸本身 为研究人员提供了令人兴奋的可能性， 研究，以及核酸进化的基本知识， 功能 修饰的核苷被用作抗病毒剂，并且具有 被用来证明新的潜力，在转录和 在翻译中引入不寻常的氨基酸。 我们的长期 目的是了解修饰核苷 化学，结构和动力学功能，特别是关于 tRNA在翻译中的作用。 有六十多个不同的 作为修饰的核苷，tRNA是最异质的生物聚合物之一 知道的 在这项研究中，修饰核苷的独特贡献 酵母tRNA（Phe）的不同局部结构及其特异性 氨酰化和反密码子-密码子识别重要性将是 测定 与核糖2 '- tRNA二级相互作用中的OH及金属离子结合特征 将阐明结构。 稳定同位素浓缩技术将 与修饰核苷和RNA化学合成相结合。 含修饰核苷的RNA序列、DNA类似物和tRNA 将产生酵母tRNA（Phe）的结构域。 他们的结构将是 通过NMR和CD光谱法测定。 我们将阐明 特异性修饰核苷对酵母tRNA（Phe）重要性 酵母苯丙氨酰-tRNA合成酶（FRS）和tRNA（Phe）的识别 与密码子相互作用。 修饰核苷对FRS的重要性 tRNA（Phe）的识别将通过测定 在存在竞争性未修饰tRNA的情况下， 和选择性修饰的tRNA茎环结构域。 生化 确定tRNA（Phe）与核糖体的密码子依赖性结合将 在存在和不存在竞争性反密码子茎环的情况下进行测定 具有和不具有选择的修饰的核苷和脱氧核糖的序列 替代品 反密码子-密码子相互作用的结构和动力学 将通过CD和NMR光谱法测定。

Modified nucleosides impart unique site-specific properties to nucleic acids, yet determining their significance to structure-function relationships has proven difficult. Presumably the evolution of modified nucleosides is connected with their altering of RNA chemistry and structure, and the potential to accomplish this is as varied as are the chemistries and structures of the modifications. An understanding of the unique chemistry, structure and dynamics that the more than 80 naturally occurring modified nucleosides contribute to nucleic acids would itself provide investigators with exciting possibilities for nucleic acid research, as well as basic knowledge of nucleic acid evolution and function. Modified nucleosides are used as anti-viral agents, and have been used to demonstrate new potentials in transcription and the introduction of unusual amino acids in translation. Our long-term objective is to understand the relationship of modified nucleoside chemistry, structure and dynamics to function, particularly with regard to tRNA function in translation. With more than sixty differently modified nucleosides, tRNA is one of the most heterogeneous bio-polymers known. In this study the unique contributions of modified nucleosides to different local structures in yeast tRNA(Phe) and their specific importance to aminoacylation and anticodon-codon recognition will be determined. The importance of modification in relation to the ribose 2'- OH in secondary interactions and metal ion binding features of tRNA structure will be elucidated. Stable isotope enrichment techniques will be combined with modified nucleoside and RNA chemical syntheses. Modified nucleoside-containing RNA sequences, DNA analogues, and tRNA domains of yeast tRNA(Phe) will be produced. Their structures will be determined by NMR and CD spectrometry. We will elucidate the biological importance of specific modified nucleosides to yeast tRNA(Phe) recognition by yeast phenylalanyl-tRNA synthetase (FRS) and tRNA(Phe) interaction with codon. The importance of modified nucleosides to FRS recognition of tRNA(Phe) will be assayed by determining the kinetics of aminoacylation of the mature tRNA in the presence of competing unmodified and selectively modified tRNA stem-loop domains. Biochemical determinations of codon-dependent binding of tRNA(Phe) to ribosomes will be assayed in the presence and absence of competing anticodon stem-loop sequences with and without selected modified nucleosides and deoxyribose substitutions. The structure and dynamics of anticodon-codon interaction will be determined by CD and NMR spectrometry.