AMINOACYL TRNA SYNTHETASE SUBTRATE RECOGNITION

氨酰 TRNA 合成酶底物识别

• 批准号: 2444412
• 负责人: Karin M Musier-Forsyth
• 金额: $ 6.84万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444412
• 关键词: DNA footprinting; Escherichia coli; RNA biosynthesis; acylation; aminoacyl tRNA; bacterial genetics; enzyme substrate; molecular site; oligonucleotides; plasmids; proline; site directed mutagenesis; transfer RNA

My goal is to establish a vigorous, independent research program that focuses on the investigation of RNA-protein interactions and structure- function relationships in biological systems. The major function of transfer RNA (tRNA) molecules is to serve as a site of attachment for amino acids, which are then activated for use in protein synthesis. The high degree of specificity between tRNAs and aminoacyl-tRNA synthetases, the enzymes that catalyze this attachment, make these molecules excellent targets for studying the principles of protein-RNA interaction. Transfer RNA molecules are also used as primers by reverse transcriptases. These are enzymes in retroviruses that function to transcribe DNA from the retroviral RNA genome. A future direction of my lab will be to understand the interaction between HIV reverse transcriptase and human tRNA(Lys), the specific RNA primer for this viral enzyme. Briefly, the primary objective of this research is to address the question of how proline tRNA synthetase (ProRS) recognizes and interacts with its cognate tRNA substrate. We have already made progress in identifying the subset of nucleotides that mark a tRNA molecule for specific aminoacylation with proline. We have also demonstrated that a functional tRNA(Pro) molecule can be assembled by annealing together two oligonucleotides representing 3'- and 5'-fragments. The use of these "annealed" substrates, prepared with a chemically-synthesized fragment, has facilitated the identification of RNA structural characteristics that are important for aminoacylation by ProRS. Novel applications of chemical RNA synthesis will enable the attachment of covalent probes at specific, internal positions of annealed tRNA substrates for use in cross-linking and fluorescence studies. In this way, protein structural motifs and specific amino acid residues in close contact with specific regions of tRNA(Pro) will be identified.

我的目标是建立一个有力的独立研究计划 重点是研究RNA - 蛋白质相互作用和结构 - 生物系统中的功能关系。主要功能 转移RNA（tRNA）分子是作为附着位点的 然后将其激活用于蛋白质合成。这 TRNA和氨基酰基-TRNA合成酶之间的高度特异性， 催化这种附着的酶，使这些分子使这些分子 研究蛋白质-RNA原理的出色目标 相互作用。通过反向转移RNA分子也用作引物 转录酶。这些是逆转录病毒中的酶 从逆转录病毒RNA基因组中转录DNA。 一个未来的方向 我的实验室将了解艾滋病毒反向之间的相互作用 转录酶和人tRNA（LYS），这是特定的RNA底漆 病毒酶。 简而言之，这项研究的主要目的是解决 脯氨酸tRNA合成酶（pror）如何识别和相互作用的问题 其同源tRNA底物。我们已经取得了进步 识别标记tRNA分子的核苷酸的子集 脯氨酸的特定氨基酰化。我们还证明了 可以通过退火一起将功能性tRNA（Pro）分子组装在一起 代表3'-和5'Fragments的寡核苷酸。这些使用 用化学合成的片段制备的“退火”底物， 促进了RNA结构特征的识别 这对于prors氨基酰化很重要。新颖的应用 化学RNA合成将使共价探针的附着 将退火tRNA底物的特定内部位置用于 交联和荧光研究。这样，蛋白质结构 基序和特定的氨基酸残基与特定 将确定tRNA区域（Pro）。