AMINOACYL TRNA SYNTHETASE SUBTRATE RECOGNITION

我的目标是建立一个充满活力的独立研究项目， 重点研究RNA-蛋白质的相互作用和结构- 生物系统中的功能关系。的主要功能 转移RNA(TRNA)分子是作为连接部位的 氨基酸，然后被激活以用于蛋白质合成。这个 TRNA和氨酰-tRNA合成酶之间的高度特异性， 催化这种连接的酶，使这些分子 蛋白质-RNA原理研究的极佳靶点 互动。转移的rna分子也被用来作为反向引物。 转录酶。这些是逆转录病毒中的酶，其功能是 从逆转录病毒RNA基因组转录DNA。未来的发展方向 我的实验室将了解艾滋病毒逆转之间的相互作用 转录酶和人tRNA(Lys)，这是该基因的特异性RNA引物 病毒酵素。 简而言之，这项研究的主要目标是解决 关于脯氨酸tRNA合成酶(ProRS)如何识别和相互作用的问题 与其同源tRNA底物。我们已经在以下方面取得进展 识别标记tRNA分子的核苷酸子集 与脯氨酸发生特异的氨酰化反应。我们还展示了一个 功能性tRNA(Pro)分子可以通过两种不同的方式组合在一起 代表3‘和5’片段的寡核苷酸。这些产品的使用 用化学合成的片断制备的“退火”衬底， 促进了对RNA结构特征的鉴定 这对ProRS的氨酰化反应是重要的。的新应用 化学RNA合成将使共价探针能够在 退火法tRNA底物的特定内部位置 交联剂和荧光研究。通过这种方式，蛋白质结构 与特定氨基酸密切接触的基序和特定氨基酸残基 将确定tRNA(Pro)的区域。

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.