TRANSFER RNA STRUCTURE DURING PROTEIN SYNTHESIS

基因表达对正常但变化的生理状态的反应 对疾病状态具有必要的核酸结构识别能力 宿主蛋白质和结果的正常和正确或异常和 蛋白质合成不正确。核酸相互作用的能力 在特定的和成功的酶反应中有望 依赖于核酸的柔顺构象识别 三维蛋白质。因此，我们需要了解的不仅仅是原子核 不仅具有酸性结构，还具有结构灵活性和运动能力， 尤其是内部运动，它对催化机理很重要 或受体激活。这些调查领域对以下方面具有重要意义 对基因表达调控的基本理解， RNA的转录和转录后加工，以及密码子 翻译的认可度和忠诚度。原生的三维 核酸在溶液中的结构、整体和特定部位的动力学 基本上是未知的。这项研究计划是以下项目的共同努力 两个实验室研究母语迁移的结构动力学 单独存在于溶液中以及与各种蛋白质相互作用时的RNA。 碳-13和质子核磁共振(核磁共振)光谱将 用来确定tRNA的整体和特定的位点动态 结构。转移RNA的甲基基团将富含13C， 在体内的其他各种位置，并提纯特定的tRNA物种。 横向和纵向弛豫(T1，T2)的~(13)C核磁共振研究 然后，NOE将在这些tRNA上以75.5 MHz(13C)的速度在 各种情况。氨基酰化和环氧氯丙烷的结构和动力学 未经酰化的物种将被比较。~(13)C-甲基富集物的相互作用 含有纯化的同源和非同源氨基酰基-tRNA的tRNA物种 合成酶和伸长因子将通过13C-核磁共振进行研究。类比 未标记的tRNA物种将通过1H-核磁共振进行研究。以帮助发出信号 高场质子共振~(13)C-甲基富集型tRNA的归属 物种将通过~(13)C偶联和去偶联的~1H-核磁共振进行研究。钥匙 ~(13)C-富集方法学的优点是能够使 明确的信号分配和评估自然结构 生理条件下使用非破坏性、非摄动 核磁共振技术。

Response of gene expression to normal yet changing physiological states and to disease states has requisite recognition of nucleic acid structure by a host of proteins and results in the normal and correct or abnormal and incorrect synthesis of proteins. The ability of nucleic acids to interact specifically and successfully in enzymatic reactions would be expected to depend on pliant conformational recognition of the nucleic acid by the protein in three dimensions. Thus, we need to understand not only nucleic acid structure but also structural flexibility and motional capability, particularly internal motion which is important for mechanisms of catalysis or receptor activation. These areas of investigation are significant to the fundamental understanding of the regulation of gene expression, transcription and post transcriptional processing of RNAs, and to codon recognition and fidelity of translation. The native three-dimensional structure, overall and site specific dynamics of nucleic acids in solution are essentially unknown. This research program is the combined effort of two laboratories to investigate the structural dynamics of native transfer RNA in solution alone and during interaction with various proteins. Carbon-13 and proton nuclear magnetic resonance (NMR) spectrometry will be used to ascertain overall and specific site dynamics of the tRNA structure. Transfer RNA will be 13C-enriched in its methyl groups and at various other locations in vivo, and specific tRNA species purified. Carbon-13 NMR studies of transverse and longitudinal relaxation (T1, T2) and NOE will then be accomplished on these tRNAs at 75.5 MHz (13C) under various conditions. The structure and dynamics of amino-acylated and unacylated species will be compared. Interaction of 13C-methyl enriched tRNA species with purified cognate and non-cognate aminoacyl-tRNA synthetases and elongation factor will be studied by 13C-NMR. Analogous unlabeled tRNA species will be studied by 1H-NMR. To aid in making signal assignments of high field proton resonances, 13C-methyl enriched tRNA species will be studied by 13C-coupled and decoupled 1H-NMR. Key advantages of the methodology of 13C-enrichment are the ability to make unequivocal signal assignments and to assess native structure under physiological conditions using the non-destructive, non-perturbing technology of NMR.