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， 主机的蛋白质和结果在正常和正确或异常， 蛋白质的错误合成。 核酸相互作用 特别是在酶促反应中成功地应用， 依赖于核酸分子对核酸的柔顺构象识别， 蛋白质的三维结构 因此，我们不仅需要了解核 酸结构，而且结构灵活性和运动能力， 特别是内部运动，这对催化机制很重要， 或受体激活。 这些调查领域对于 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 翻译的识别性和忠实性。 原生三维 溶液中核酸结构、整体和位点特异性动力学 基本上是未知的。 这项研究计划是联合努力， 两个实验室，研究自然转移的结构动力学 RNA单独存在于溶液中以及与各种蛋白质相互作用期间。 碳-13和质子核磁共振（NMR）光谱将被用于 用于确定tRNA的总体和特定位点动力学 结构 转移RNA将在其甲基中富集13 C，并且在 在体内的各种其他位置，以及纯化的特异性tRNA种类。 横向和纵向弛豫（T1，T2）的碳-13 NMR研究 然后在75.5 MHz（13 C）下在这些tRNA上完成NOE。 各种条件。 氨基酰化和 将比较未酰化的物质。 富集13 C-甲基的相互作用 具有纯化的同源和非同源氨酰-tRNA的tRNA物质 合成酶和延伸因子将通过13 C-NMR进行研究。 类似 将通过1H-NMR研究未标记的tRNA种类。 帮助发出信号 高场质子共振分配，13 C-甲基富集的tRNA 将通过13 C-偶联和去偶联的1H-NMR研究物种。 关键 13 C富集方法的优点是能够使 明确的信号分配和评估天然结构， 使用非破坏性的、非干扰性的 NMR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.