Modified Uridines, Contributors of Novel Chemistries to Functional RNA Structures

小行星9631103 该项目有两个总体目标。第一个目标是确定化学/结构/功能的关系，选定的修改尿苷，具有特定的，但无法解释的，生物相关性。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，设计和产生具有某些特征的RNA结构。 具体目标是：确定mnm 5s 2U 34和mcm 5s 2U 34的物理化学性质，这些性质有助于tRNALys中生物学上重要的和可能非典型的反密码子构象。初步数据表明，修饰的尿苷与N6-苏氨酰氨基甲酰基腺苷-37（t6 A37）相互作用，即使在像五核苷酸一样小的模型系统中也会产生一种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以解释关联tRNA的赖氨酰-和/或谷氨酰-tRNA合成酶识别以及在密码子阅读中观察到的tRNALys行为。利用从tRNA和模型系统的研究中获得的信息，将设计和合成一种修饰的核苷依赖性相互作用，从而产生一种新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Mg 2+的结合。tRNA与Mg ~（2+）的结合对于其功能是重要的。氨基酸修饰的尿苷和腺苷结合金属，并且二氢尿苷（D）在tRNA的D环和额外臂中的定位与亲水性金属结合尿苷3- 3-（S）-氨基-3-羧基丙基-尿苷（acp 3U）的出现相关。初步数据表明，D几乎是100%的2 '-内切，并且它在3'-相邻核苷中诱导2 '-内切折叠。tRNA的D和TpsC结构域的组合产生高亲和力Mg+结合位点。然而，有太少的信息来预测哪些结构构成RNA中的高亲和力Mg 2+结合位点，一般来说，特别是tRNA。了解氨基酸修饰核苷的金属螯合作用和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，将根据本研究中修饰核苷的化学和结构贡献的知识设计和合成修饰核苷依赖性高亲和力金属结合RNA。 修饰的尿苷和t6 A将使用手动和自动化学合成引入小（3- 6聚体）和大（17- 19聚体）天然存在的序列中。将生产足量的低聚物，以使用紫外和圆二色谱法确认金属离子和溶液条件对化学和结构影响的初步证据。将通过分析酶活性和凝胶位移来评估与氨酰-tRNA合成酶的相互作用。与核糖体的功能性相互作用将通过抑制密码子依赖性tRNA结合和化学修饰来检测。选定的结构，通过NMR分析，将与分子动力学细化，NMR推导的距离和扭转角约束建模。计算机辅助设计将导致新的修饰的尿苷，假环状和Mg 2+结合RNA的合成和分析。 DNA中编码的是生产蛋白质的遗传信息。 转移核糖核酸，转移核糖核酸，将遗传信息解码成每个新蛋白质中正确的氨基酸序列。 转运蛋白由腺苷、鸟苷、胞苷和尿苷四种核苷组成，这四种核苷有少量的修饰。 修饰的尿苷是特别感兴趣的，因为它们出现在tRNA中的位置，在那里它们有助于正确的阅读和翻译遗传密码的进展。 该项目有两个目标。 由于修饰的尿苷可能影响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的尿苷但负责将两种不同的氨基酸（谷氨酸和赖氨酸）带到蛋白质制造单元（核糖体）的tRNA的解码结构。 修饰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。 在细菌和哺乳动物细胞中，谷氨酸和溶素tRNA的能力负责无法解释的生物学观察。 ***

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. ***