MODIFIED NUCLEOSIDE STRUCTURE FUNCTION RELATIONS--TRNA

修饰的核苷赋予核糖核酸独特的位点特异性 酸，但确定它们对结构功能的意义 事实证明，两人的关系很困难。大概是修饰语的进化 核苷与其改变RNA化学和 结构，实现这一目标的潜力是多种多样的 修饰的化学成分和结构。对这一概念的理解 独特的化学、结构和动力学，使80多种天然 发生修饰的核苷对核酸本身有贡献 为研究人员提供了令人兴奋的核酸可能性 研究，以及核酸进化和 功能。修饰的核苷被用作抗病毒药物，并已 被用来展示转录的新潜力，以及 在翻译中引入不寻常的氨基酸。我们的长期合作 目的是了解修饰后的核苷与蛋白质的关系 化学、结构和动力学的作用，特别是在 目的：tRNA在翻译中的作用。有六十多种不同的 修饰的核苷，tRNA是最不均匀的生物聚合物之一。 为人所知。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 对氨基酰化和反密码子识别的重要性将是 下定决心。与核糖2‘-相关的修饰的重要性 TRNA二级相互作用中的OH及金属离子结合特性 结构将会被阐明。稳定的同位素浓缩技术将 与修饰的核苷和RNA化学合成相结合。 修饰的含有核苷的RNA序列、DNA类似物和tRNA 将产生酵母tRNA结构域(Phe)。他们的结构将是 经核磁共振和圆二色谱测定。我们将阐明生物学上的 特定修饰核苷对酵母tRNA(Phe)的重要性 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。修饰核苷在FRS中的重要性 TRNA(Phe)的识别将通过测定 竞争未修饰条件下成熟tRNA的氨酰化反应 和选择性修饰的tRNA茎环结构域。生化 依赖密码子的tRNA(Phe)与核糖体结合的测定将 在存在和不存在竞争反密码子茎环的情况下进行检测 含和不含所选修饰核苷和脱氧核糖的序列 替换。反密码子-密码子相互作用的结构和动力学 将通过CD和核磁共振光谱分析来确定。

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.