STRUCTURAL BASIS OF TRANSFER RIBONUCLEIC ACID FUNCTION

将使用高分辨率19F核磁共振(NMR)来探测 5-氟尿嘧啶(Fura)取代的大肠杆菌的溶液结构 ColitRNA。5-氟尿苷以生物合成的方式结合到tRNA中， 取代尿苷和尿苷衍生的次碱，完全生成 含有高度敏感的局部探针的功能分子。这些是 分布在tRNA的螺旋区和环区，并可以 作为分子所有部分结构变化的报告者。 纯化了三个含氟核糖核酸，得到了~(19)F-核磁共振波谱 在282 MHz下，几乎可以分解所有结合的Fura残留物。分配给 19F共振将允许氨基酰化的影响，并且 TRNA与阳离子、蛋白质等的相互作用有待研究 翻译过来了。高峰分配已经开始，这项工作将 继续，利用几种方法，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，以帮助解释 含Fura的合成寡核苷酸和多核苷酸的~(19)F核磁共振波谱 将准备不同的序列上下文。化学位移和 螺旋结构和无规线圈中Fura残基的松弛将 被检查以确定二级结构的影响，邻近 富拉残基，螺旋的磨损，以及相邻碱基的性质。 同源和非同源氨基酰-tRNA合成酶的特异性相互作用 还将用19F核磁共振检查tRNA，以测试拟议的形成 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。因为尿苷衍生的次碱是 被Fura取代的修饰碱基在蛋白质启动中的作用 还将研究合成和密码子识别。 与大多数其他tRNAs不同，富拉取代的大肠杆菌tRNAys不是很 活跃于蛋白质合成。此tRNA将被提纯，并且结构 将对不活动的基础进行调查。最近的证据表明 Fura的抗肿瘤作用及其细胞毒性可能在一定程度上被利用 将Fura整合到细胞RNA中。一项关于以下问题的研究 Fura掺入细菌的RNA中，在其中发生掺入 比在哺乳动物细胞中更容易获得，应该为 了解氟代嘧啶的作用。

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.