Modified Uridines, Contributors of Novel Chemistries to Functional RNA Structures

9631103农业发展项目有两个总体目标。第一个目标是确定选定的具有特定的、但尚未解释的生物相关性的修饰尿酸的化学/结构/功能关系。在第二个目标中，解释这些生物观察的修饰核苷的化学和结构贡献将被用于预测、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，它们对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有贡献。初步数据表明，修饰的尿苷与N6-苏氨酰氨基甲酰腺苷-37(T6A37)相互作用，即使在五核苷酸这样小的模型系统中也会产生一个新的、非共价产生的环。反密码子结构域中的这种独特结构可能解释了赖氨酰和/或谷氨酰tRNA合成酶对同源tRNAs的识别以及tRNAys在密码子阅读中的观察到的行为。利用tRNAs和模型系统的研究信息，将设计和合成一种改进的依赖于核苷的相互作用，从而产生一种新型的共价键合的假环状RNA。(Ii)。确定修饰的尿苷和腺苷的金属结合特性是否有助于tRNA与镁离子的结合。TRNA与镁离子的结合是发挥作用的重要因素。氨基酸修饰的尿苷和腺苷与金属结合，二氢尿苷(D)在tRNA环和额外臂上的定位与亲水性金属结合尿苷3-3-(S)-氨基-3-羧丙基尿苷(Acp3U)的出现有关。初步数据表明，D几乎是100%的2‘-内切，它能在3’-邻位核苷中诱导2‘-内切。TRNA的D和TpsC结构域的结合产生了一个高亲和力的镁离子结合部位。然而，通常情况下，预测RNA，特别是TRNA中哪些结构构成高亲和力的镁结合位点的信息太少。了解氨基酸修饰的核苷对金属的络合作用以及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知识，设计和合成一种修饰的依赖于核苷的高亲和力金属结合RNA。修饰的尿苷和t6A将通过人工和自动的化学合成被引入到小的(3-6个聚体)和大的(17-19个聚体)自然产生的序列中。将产生足够数量的低聚物，以利用紫外线和圆二色谱确认金属离子和溶液条件对化学和结构影响的初步证据。与氨酰-tRNA合成酶的相互作用将通过酶活性和凝胶位移的分析来评估。与核糖体的功能相互作用将通过抑制依赖于密码子的tRNA结合和化学修饰来检测。选定的结构，通过核磁共振分析，将通过分子动力学精炼、核磁共振导出的距离和扭角约束进行建模。计算机辅助设计将导致合成和分析新型修饰的含尿苷、假环状和结合镁离子的RNA。编码在DNA中的%是生产蛋白质的遗传信息。转移核糖核酸，tRNAs，将这种遗传信息解码为每种新蛋白质中正确的氨基酸序列。TRNAs由四种核苷组成，即腺苷、鸟苷、胞苷和尿苷，以及这四种核苷的修饰。修饰的尿氨酸特别令人感兴趣，因为它们出现在tRNA中有助于正确阅读和翻译遗传密码的位置。该项目有两个目标。由于修饰的尿氨酸可能会影响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到蛋白质制造单位核糖体的tRNA的解码结构。修饰后的尿酸可能结合金属离子，如镁离子，从而影响读码结构和功能。谷氨酸和裂解酶tRNAs的能力是细菌和哺乳动物细胞中无法解释的生物学观察的原因。***

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. ***