Modified Uridines, Contributors of Novel Chemistries to Functional RNA Structures

修饰尿苷，功能性 RNA 结构新化学的贡献者

• 批准号: 9986011
• 负责人: Paul Agris
• 金额: $ 37.01万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-15 至 2004-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9986011&HistoricalAwards=false
• 关键词: Modified; Uridines; Contributors; Novel; Chemistries

9986011AgrisRNAs contain the standard nucleosides adenosine, uridine, guanosine and cytosine. In addition, RNA contains some 100 naturally occurring modifications of the four major nucleosides. The modifications are as simple as methylations and thiolations and as complex as amino acid derivatives and tricylic additions. All are enzymatically synthesized after synthesis (transcription) of the RNA. Transfer RNAs (tRNA) having a large number and variety of modified nucleosides, its structure and function readily assayed, is a good model RNA for the study of modified nucleoside contributions to structure and function. Conventional wisdom has led researchers to believe that tRNA, responsible for bringing individual amino acids to the ribosome in response to genetic coding triplets (codons) in the messenger RNA (mRNA), do not require modified nucleosides to function in protein synthesis. However, this laboratory has recently determined that at least two modified nucleosides, 2-thiouridine derivatives (s2U) at "wobble" position-34 of and 6-threonylcarbamoyladenosine (t6A) at position-37 of tRNA's anticodon stem and loop domain (ASL), are individually capable of restoring ribosome binding to the otherwise inactive ASL of human lysine-3 tRNA. Position-34 is the first nucleoside of the anticodon that binds its complementary codon in the mRNA on the ribosome. Thiouridines occur in tRNAs for glutamine, glutamic acid and lysine; t6A occurs in tRNAs for lysine. Techniques for site-specific introduction of modified and stable isotope labeled nucleosides developed in this laboratory make it possible to probe the contributions these nucleosides alone and together, and in combination with other modifications provide to RNA function and structure. The project's long-term objective continues to be an elucidation of the physicochemical contributions of modified uridines to the biological functions of RNA. Several testable hypotheses have been formulated: a) Thiouridine-34 dependent ribosome binding is common to tRNAs with uridine-rich anticodon loops and is important for A-site, as well as P-site binding. b) Whereas t6A37 restores ribosomal binding to the ASLLys3UUU, the commonly occurring position-37 modification of glutamine and glutamic acid tRNAs, 2-methyladenosine-37 (m2A37), will not restore binding to ASLGlnSUC and ASLGluSCU. c) Modification-dependent ribosome binding is sequence context dependent. d) Anticodon stem and loop modifications provide optimum ribosomal binding architecture to lysine, glutamine and glutamic acid tRNAs by significantly influencing chemical and conformational dynamics of the ASL. Biochemical and molecular biology techniques will be used as approaches, including the use of quantitative P- and A-site ribosomal binding, and quantitative footprinting of tRNA on ribosomal P- and A-site 16S rRNA nucleosides. The physicochemical contributions of modifications (thermal stability, base stacking interactions and structure) will be related to their abilities to restore ribosome binding to lysine, glutamine and glutamic acid ASLs. Effects of modification on ribosome binding and footprinting will be related to the modifications' effects on stability, structure and dynamics.

9986011 AgrisRNA含有标准核苷腺苷、尿苷、鸟苷和胞嘧啶。 此外，RNA含有约100种天然存在的四种主要核苷修饰。 修饰简单如甲基化和巯基化，复杂如氨基酸衍生物和三环加成。所有这些都是在RNA合成（转录）后酶促合成的。转运RNA（transferRNA，tRNA）具有大量的核苷修饰，其结构和功能易于分析，是研究核苷修饰对结构和功能影响的理想模型RNA。传统观点使研究人员相信，负责将单个氨基酸带到核糖体以响应信使RNA（mRNA）中的遗传编码三联体（密码子）的tRNA，不需要修饰的核苷就能在蛋白质合成中发挥作用。 然而，该实验室最近已经确定，至少两种修饰的核苷，位于tRNA的反密码子茎环结构域（ASL）的“摆动”位置-34的2-硫代尿苷衍生物（s2 U）和位于位置-37的6-苏氨酰氨基甲酰基腺苷（t6 A），能够单独地恢复核糖体与人赖氨酸-3 tRNA的否则无活性的ASL的结合。 位置-34是反密码子的第一个核苷，其结合核糖体上mRNA中的互补密码子。 硫尿苷存在于谷氨酰胺、谷氨酸和赖氨酸的tRNA中; t6 A存在于赖氨酸的tRNA中。 在本实验室开发的用于位点特异性引入经修饰的和稳定的同位素标记的核苷的技术使得可以探测这些核苷单独和一起以及与其他修饰组合提供的对RNA功能和结构的贡献。该项目的长期目标仍然是阐明修饰的尿苷对RNA生物学功能的物理化学贡献。已经提出了几个可检验的假设： a）硫尿苷-34依赖性核糖体结合对于具有富含尿苷的反密码子环的tRNA是常见的，并且对于A位点以及P位点结合是重要的。B）尽管t6 A37恢复了核糖体与ASLLys 3 UUU的结合，但谷氨酰胺和谷氨酸tRNA的通常发生的位置-37修饰，2-甲基腺苷-37（m2 A37），将不会恢复与ASLGlnSUC和ASLGluSCU的结合。c）修饰依赖性核糖体结合是序列环境依赖性的。 d）反密码子茎和环修饰通过显著影响ASL的化学和构象动力学，提供与赖氨酸、谷氨酰胺和谷氨酸tRNA的最佳核糖体结合结构。将使用生物化学和分子生物学技术作为方法，包括使用定量P-和A-位点核糖体结合，以及核糖体P-和A-位点16 S rRNA核苷上tRNA的定量足迹。修饰的物理化学贡献（热稳定性、碱基堆积相互作用和结构）将与其恢复核糖体与赖氨酸、谷氨酰胺和谷氨酸ASL结合的能力相关。 修饰对核糖体结合和足迹的影响将与修饰对稳定性、结构和动力学的影响有关。