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: 9631103
• 负责人: Paul Agris
• 金额: $ 36.8万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9631103&HistoricalAwards=false
• 关键词: Modified; Uridines; Contributors; Novel; Chemistries

9631103 Agris The project has two overall objectives. The first objective is to determine the chemistry/structure/ function relationships of selected modified uridines that have specific, yet unexplained, biological relevance. In the second objective, the chemical and structural contributions of modified nucleosides that explain these biological observations will be used to predict, design and produce RNA structures with certain characteristics. The specific aims are to: (i). Determine the physicochemical properties of mnm5s2U34 and mcm5s2U34 that contribute to a biologically important and possibly atypical anticodon conformation in tRNALys. Preliminary data indicate that the modified uridines interact with N6-threonylcarbamoyladenosine-37 (t6A37) creating a novel, non-covalently produced loop even in as small a model system as a pentanucleotide. This unique structure within the anticodon domain may explain both lysyl- and/or glutamyl-tRNA synthetase recognition of cognate tRNAs and the observed behavior of tRNALys in codon reading. Using information from the study of tRNAs and model systems, a modified nucleoside-dependent interaction resulting in a novel covalently bonded, pseudo-circular RNA will be designed and synthesized. (ii). Determine if the metal-binding properties of modified uridines and adenosines contribute to tRNA's binding of Mg2+. tRNA's binding of Mg2+ is important to function. Amino acid modified uridines and adenosines bind metals and the localization of dihydrouridine (D) in Dloops and extra arms of tRNAs correlates with the appearance of the hydrophilic, metal-binding uridine, 3- 3-(S)-amino-3-carboxypropyl -uridine (acp3U). Preliminary data indicate that D is almost 100% 2'-endo and that it induces 2'-endo pucker in 3'-adjacent nucleosides. The combination of the D and TpsC domains of tRNA produces a high affinity Mg+ binding site. However, there is too little information to predict which structures constitute high affinity Mg2+ binding sites in RNA, in general, and tR NAs in particular. An understanding of metal chelation by amino acid modified nucleosides and the influence of D on local structure will aid in understanding the functional significance of metal binding. As part of this specific aim, a modified nucleoside-dependent, high affinity metal binding RNA will be designed and synthesized based on knowledge of the chemical and structural contributions of the modified nucleosides in this study. Modified uridines and t6A will be introduced into small (3-6mers) and large (17-19mers) naturally-occurring sequences using manual and automated chemical syntheses. Sufficient quantities of oligomers will be produced to confirm, using ultraviolet and circular dichroism spectroscopy, preliminary evidence of the effects of metal ions and solution conditions on chemistry and structure. Interactions with aminoacyl-tRNA synthetase will be assessed by analysis of enzyme activity and gel shifts. Functional interactions with the ribosome will be detected by inhibition of codon-dependent tRNA binding and chemical modification. Selected structures, analyzed by NMR, will be modeled with molecular dynamics refined, NMR derived distance and torsion angle constraints. Computer-assisted design will lead to the synthesis and analysis of novel modified uridine-containing, pseudo-circular and Mg2+ binding RNAs. %%% Encoded within DNA is genetic information for the production of proteins. Transfer ribonucelic acids, tRNAs, decode this gentic information into the correct sequence of amino acids in each new protein. tRNAs are composed of four nucleosides, adenosine, guanosine, cytidine anduridine, and minot amoounts of modifications of these four. Modified uridines are of particular interest because they occur at positions in tRNA where they contribute to the correct reading and progress in translating the genetic code. The project has two objectives. Since modified uridines may affect tRNA structure, and thus tRNA's ability to decode, the decoding structures of tRNAs that have the same modified uridin, but are responsible for bringing two different amino acids, glutamic acid and lysine, to the protein manufacturing unit, the ribosome, will be determined. Modified uridines may bind metal ions, such as magnesium, and affect code-reading strucute and function. The ability of glutamic acid and lysin tRNAs are responsible for unexplained biological observations in both bacterial and mammalian cells. ***

9631103 Agris项目有两个总体目标。第一个目标是确定选定的具有特定但尚未解释的生物学相关性的改性尿苷的化学/结构/功能关系。在第二个目标中，解释这些生物学观察的修饰核苷的化学和结构贡献将用于预测、设计和生产具有某些特征的RNA结构。具体目标是：(i)。确定mnm5s2U34和mcm5s2U34的物理化学性质，这些性质有助于tRNALys中具有重要生物学意义且可能是非典型的反密码子构象。初步数据表明，修饰的尿苷与n6 -苏酰基氨基腺苷-37 （t6A37）相互作用，即使在像五核苷酸这样小的模型系统中也能产生新的非共价产生的环。反密码子结构域内的这种独特结构可以解释赖氨酸和/或谷氨酰胺- trna合成酶对同源trna的识别以及tRNALys在密码子读取中观察到的行为。利用trna和模型系统研究的信息，修饰核苷依赖相互作用导致新的共价键，伪环状RNA将被设计和合成。(二)。确定修饰的尿苷和腺苷的金属结合特性是否有助于tRNA与Mg2+的结合。tRNA与Mg2+的结合对功能至关重要。氨基酸修饰的尿苷和腺苷结合金属，二氢尿苷(D)在trna的Dloops和额外臂上的定位与亲水性、金属结合的尿苷3- 3-(S)-氨基-3-羧基丙基尿苷（acp3U）的出现相关。初步数据表明，D几乎100%是2'-末端，并且在3'-邻近核苷中诱导2'-末端卷曲。tRNA的D和TpsC结构域结合产生高亲和力的Mg+结合位点。然而，一般来说，预测哪些结构构成RNA中高亲和力的Mg2+结合位点的信息太少，特别是tR NAs。了解氨基酸修饰核苷的金属螯合作用以及D对局部结构的影响将有助于理解金属结合的功能意义。作为这一特定目标的一部分，本研究将基于对修饰核苷的化学和结构贡献的了解，设计和合成一种修饰的核苷依赖性高亲和力金属结合RNA。通过人工和自动化学合成，将修饰的尿嘧啶和t6A引入小（3-6mers）和大（17-19mers）天然序列中。将生产足够数量的低聚物，利用紫外线和圆二色光谱来证实金属离子和溶液条件对化学和结构的影响的初步证据。与氨基酰基- trna合成酶的相互作用将通过酶活性分析和凝胶转移来评估。与核糖体的功能相互作用将通过抑制密码子依赖的tRNA结合和化学修饰来检测。选定的结构，通过核磁共振分析，将与分子动力学细化，核磁共振导出的距离和扭转角约束建模。计算机辅助设计将导致合成和分析新的修饰的含尿嘧啶rna，伪环状rna和Mg2+结合rna。编码在DNA内的是生产蛋白质的遗传信息。将核糖核酸，trna，解码成每个新蛋白质中正确的氨基酸序列的遗传信息。trna由四种核苷组成：腺苷、鸟苷、胞苷和尿苷，以及这四种核苷的少量修饰。修饰的尿苷是特别有趣的，因为它们出现在tRNA的位置，在那里它们有助于正确阅读和翻译遗传密码。该项目有两个目标。由于修饰的尿苷可能会影响tRNA的结构，从而影响tRNA的解码能力，因此将确定具有相同修饰尿苷但负责将两种不同的氨基酸（谷氨酸和赖氨酸）带入蛋白质制造单元核糖体的tRNA的解码结构。修改后的尿苷可以结合金属离子，如镁，并影响代码读取结构和功能。谷氨酸和溶酶trna的能力是细菌和哺乳动物细胞中无法解释的生物学观察结果的原因。＊＊＊