Modified Uridines, Contributors of Novel Chemistries to Functional RNA Structures

9986011农业rna含有标准核苷腺苷、尿苷、鸟苷和胞嘧啶。此外，RNA含有大约100种自然发生的四种主要核苷的修饰。这些修饰像甲基化和硫代化一样简单，像氨基酸衍生物和三环添加一样复杂。它们都是在RNA合成（转录）后酶促合成的。转移RNA （Transfer RNA, tRNA）具有数量众多、种类繁多的修饰核苷，其结构和功能易于测定，是研究修饰核苷对结构和功能贡献的良好模型RNA。传统观点使研究人员相信，tRNA负责将单个氨基酸带到核糖体中，以响应信使RNA （mRNA）中的遗传编码三联体（密码子），而不需要修饰的核苷来发挥蛋白质合成的作用。然而，该实验室最近确定了至少两种修饰的核苷，位于tRNA的反密码子干环结构域（ASL）的“wobble”位置-34的2-硫脲衍生物（s2U）和位于反密码子干环结构域（ASL）的位置-37的6-苏酰基氨基氨基腺苷（t6A），分别能够恢复核糖体与人类赖氨酸-3 tRNA的非活性ASL的结合。位置-34是反密码子的第一个核苷，它与核糖体mRNA上的互补密码子结合。硫脲存在于谷氨酰胺、谷氨酸和赖氨酸的trna中；t6A存在于赖氨酸的trna中。本实验室开发的位点特异性引入修饰和稳定同位素标记核苷的技术，使得探测这些核苷单独和一起以及与其他修饰结合对RNA功能和结构的贡献成为可能。该项目的长期目标仍然是阐明修饰尿苷对RNA生物学功能的物理化学贡献。已经提出了几个可验证的假设：a)依赖硫尿嘧啶-34的核糖体结合对于具有富尿嘧啶反密码子环的trna来说是常见的，并且对于a位点和p位点的结合都很重要。b)虽然t6A37恢复了与aslys3uuu的核糖体结合，但谷氨酰胺和谷氨酸trna的37位修饰，2-甲基腺苷-37 （m2A37）不会恢复与ASLGlnSUC和ASLGluSCU的结合。c)修饰依赖性核糖体结合依赖于序列上下文。d)反密码子干和环修饰通过显著影响ASL的化学和构象动力学，为赖氨酸、谷氨酰胺和谷氨酸trna提供最佳的核糖体结合结构。将使用生化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tRNA在核糖体P-和a -位点16S rRNA核苷上的定量足迹。修饰的物理化学贡献（热稳定性、碱基堆叠相互作用和结构）将与它们恢复核糖体与赖氨酸、谷氨酰胺和谷氨酸asl结合的能力有关。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。

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.