STRUCTURAL BASIS OF TRANSFER RIBONUCLEIC ACID FUNCTION

高分辨率19F核磁共振（NMR）将用于探测 5-氟尿嘧啶（FUra）取代的大肠杆菌的溶液结构 大肠杆菌 tRNA。 5-氟尿苷通过生物合成掺入 tRNA， 取代尿苷和尿苷衍生的次要碱基，完全产生 含有高度敏感的局部探针的功能分子。 这些是 分布在 tRNA 的整个螺旋区和环区，并且可以 作为分子所有部分结构变化的报告者。 纯化了三种氟化 tRNA，并获得了 19F NMR 谱 在 282 MHz 下几乎可以解析所有掺入的 FUra 残留物。 分配 19F 共振将允许氨酰化的影响，并且 tRNA与阳离子、蛋白质等的相互作用有待研究和 解释了。 高峰任务已经开始，这项工作将 继续，利用多种方法，包括分子研究 片段、化学修饰、特定区域的酶替代 tRNA、寡核苷酸结合等。帮助解释 19F NMR 谱，含有 FUra 的合成寡核苷酸和多核苷酸 将准备不同的序列上下文。 化学位移和 螺旋结构和无规卷曲中 FUra 残基的松弛将 检查以确定二级结构、邻近结构的影响 FUra 残基、螺旋磨损以及邻近碱基的性质。 同源和非同源氨酰基-tRNA 合成酶的特异性相互作用 tRNA 也将通过 19F NMR 进行检查，以测试建议的形成 酶与尿嘧啶或修饰的尿嘧啶之间的共价加合物 位于 tRNA 的第 8 位。 因为尿苷衍生的小碱基是 被FUra取代，修饰碱基在蛋白质起始中的作用 合成和密码子识别也将被研究。 与大多数其他 tRNA 不同，FUra 取代的大肠杆菌 tRNALys 不是很 参与蛋白质合成。 该 tRNA 将被纯化并且结构 将调查不活动的依据。 最近的证据表明 FUra 的抗肿瘤作用及其细胞毒性可能部分被利用 FUra 掺入细胞 RNA 中。 对后果的研究 FUra 掺入细菌的 RNA，其中发生掺入 比哺乳动物细胞更容易，应该提供一个模型 了解氟嘧啶类药物的作用。

High-resolution 19F nuclear magnetic resonance (NMR) will be used to probe the solution structure of 5-fluorouracil (FUra)-substituted Escherichia coli tRNA. 5-Fluorouridine is incorporated biosynthetically into tRNA, replacing uridine and uridine-derived minor bases, yielding fully functional molecules containing highly sensitive local probes. These are distributed throughout the helical and loop regions of the tRNA and can serve as reporters of structural changes in all parts of the molecule. Three fluorinated tRNAs have been purified and the 19F NMR spectra obtained at 282 MHz resolve virtually all incorporated FUra residues. Assignment of the 19F resonances will permit the effects of aminoacylation, and the interaction of tRNA with cations, proteins, etc. to be studied and interpreted. A start has been made on peak assignments and this work will continue, making use of several approaches including studies with molecular fragments, chemical modification, enzymatic replacement of specific regions of the tRNA, oligonucleotide binding etc. To aid in the interpretation of 19F NMR spectra, synthetic oligo- and polynucleotides containing FUra in different sequence contexts will be prepared. The chemical shifts and relaxation of FUra residues in helical structures and in random coils will be examined to determine the effects of secondary structure, neighboring FUra residues, fraying of helices, and the nature of neighboring bases. Specific interaction of cognate and non-cognate aminoacyl-tRNA synthetases with tRNA will also be examined by 19F NMR to test the proposed formation of a covalent adduct between the enzyme and the uracil or modified uracil at position 8 in the tRNA. Because uridine-derived minor bases are replaced by FUra, the role of modified bases in the initiation of protein synthesis and codon recognition will also be investigated. FUra-substituted E. coli tRNALys, unlike most other tRNAs, is not very active in protein synthesis. This tRNA will purified and the structural basis for the inactivity will be investigated. Recent evidence suggests that the antitumor effects of FUra and its cytotoxicity may in part be use to FUra incorporation into cellular RNA. A study of the consequences of FUra incorporation into the RNA of bacteria, in which incorporation occurs more readily than in mammalian cells, should provide a model for understanding the action of fluoropyrimidines.