TRANSFER RIBONUCLEIC ACID STRUCTURE AND FUNCTION

转移核糖核酸结构和功能

• 批准号: 3304963
• 负责人: JACK HOROWITZ
• 金额: $ 9.61万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304963
• 关键词: aminoacid tRNA ligase; aminoacyl tRNA; chemical kinetics; chemical structure function; chemical substitution; conformation; fluorouracil; hybrid enzyme; methyltransferase; mutant; nuclear magnetic resonance spectroscopy; nucleic acid sequence; nucleic acid structure; protein biosynthesis; site directed mutagenesis; synthetic nucleic acid; transfer RNA; transposon /insertion element; valine

The goals of this research are to define the sequence and structural elements of tRNA required for its recognition by proteins. Our particular interest is the recognition of E. coli tRNAVal by aminoacyl-tRNA synthetases and by the tRNA modifying enzyme, tRNA (uracil-5- )methyltransferase. Detailed understanding of these interactions will clarify questions concerning the fidelity of protein synthesis, the mechanisms of tRNA modification, and the role of modified nucleosides in tRNA function. The elements of tRNAVal essential for its recognition by valyl-tRNA synthetase (VRS) will be determined by examining the charging kinetics of tRNAVal variants synthesized in vitro by transcription from (cloned) mutant tRNA genes. Minimal requirements for synthetase recognition will be explored with tRNA fragments containing the essential recognition elements of tRNAVal. The structure of wild-type and mutant tRNAVal will be probed by 19F NMR spectroscopy of tRNAs labeled with fluorine by incorporation of 5-fluorouracil (FUra) in place of uracil. Fluorinated tRNAVal is ideally suited for such studies because it remains fully functional and contains highly sensitive 19F probes distributed throughout all the helical stems and loops of the tRNA molecule where they can serve to monitor local structural changes. The 19F NMR spectrum of (FUra)tRNAVal shows a resolved resonance for each of the 14 incorporated FUra residues. Now that each of these has been assigned, we plan to use two-dimensional heteronuclear NOE (HOESY) techniques to examine anticodon loop structure. 19F NMR spectroscopy will also be used to compare the interaction of (FUra)tRNAVal and mutants with cognate and non-cognate synthetases, to help clarify differences in the way these enzymes recognize tRNA. The interaction of tRNAVal with tRNA (uracil-5-)methyltransferase, will also be followed by 19F NMR, to explore conformational changes induced in the tRNA by the enzyme, and to elucidate details of the enzyme mechanisms and of the structures of covalent tRNA-protein reaction intermediates. The results of these investigations will provide basic information on protein-tRNA interactions and on the relation between tRNA structure and function. Study of the consequences of FUra incorporation into tRNA should contribute to a better understanding of the antitumor effects of fluoropyrimidines and their cytotoxicity.

这项研究的目标是确定顺序和结构 蛋白质识别所需的tRNA元件。我们的特别之处 感兴趣的是氨基酰基-tRNA识别大肠杆菌tRNAVal 合成酶和tRNA修饰酶tRNA(尿嘧啶-5- )甲基转移酶。详细了解这些交互作用将 澄清有关蛋白质合成保真度的问题， TRNA修饰的机制，以及修饰的核苷在 TRNA功能。TRNAVal对其识别所必需的元件 Valyl-tRNA合成酶(VRS)将通过检查电荷来确定 体外转录合成tRNAVal变异体的动力学研究 (克隆)突变的tRNA基因。合成酶的最低要求 将通过包含必需的tRNA片段来探索识别 TRNAVal的识别元件。野生型和突变型的结构 TRNAVal将通过标记的tRNAs的19F核磁共振波谱进行探测 用5-氟尿嘧啶(FURA)代替尿嘧啶进行氟化。 氟化的tRNAVal非常适合于这样的研究，因为它仍然 功能齐全，包含分布的高度敏感的19F探头 在tRNA分子的所有螺旋茎和环中，它们 可以用来监测当地的结构变化。~(19)F-核磁共振波谱 (Fura)tRNAVal显示了14个合并的 富拉残留物。现在这些都已经分配好了，我们计划使用 检测反密码子的二维异核NOE(HOESY)技术 循环结构。19F核磁共振波谱也将被用来比较 (Fura)tRNAVal及其突变体与同源和非同源基因的相互作用 合成酶，以帮助澄清这些酶识别方式的差异 TRNA。TRNAVal与tRNA(尿嘧啶-5-)甲基转移酶的相互作用 之后还将进行19F核磁共振，以探索引起的构象变化 通过酶在tRNA中的作用，并阐明酶的细节 共价tRNA-蛋白质反应的机理和结构 中间体。这些调查的结果将提供基本的 蛋白质-tRNA相互作用和tRNA之间关系的信息 结构和功能。富拉公司合并的后果研究 应该有助于更好地理解抗肿瘤 氟代嘧啶类化合物的作用及其细胞毒性。