Investigations of Reactions of Physiological Importance

描述(由申请人提供)： 遗传密码是在20多亿年前建立起来的。转移RNA和氨基酰tRNA合成酶是该密码的核心。合成酶催化20种氨基酸中的每一种与它们的同源tRNA相连。因为每个tRNA都含有特定氨基酸的反密码子三联体，所以氨酰化反应建立了核苷酸三联体和特定氨基酸之间的联系。转移RNA由两个主要结构域组成，这两个结构域在进化中是保守的。这些结构域以三维形式排列在L形状的结构中。一个结构域是微型螺旋，一个12个碱基对的发夹螺旋，在3‘端是单链的，包含氨基酸连接位点。第二个结构域与第一个结构域成直角，是一个茎双环，其中一个环编码反密码子三联体。这两个领域被许多人认为是独立出现的。实验的目的实际上是探索如何通过两个结构域之间的非共价相互作用将密码的三联体与连接的氨基酸连接起来，从而实现从微型螺旋到遗传密码的转变。由于氨基酰化的RNA小螺旋被认为是氨基酰tRNAs的可能前体，第二个目标是建立基于小螺旋的多肽合成系统。这些系统旨在说明至少几种从假定的RNA世界过渡到蛋白质剧场的方法。它们还重现了当代蛋白质合成的一些特征，包括依赖于RNA模板的反应的可能性。第三个目标是理解依赖于RNA的氨基酸区别，这使得现代通用代码成为可能。具体地说，实验旨在阐明tRNAs中的微妙信息如何与合成酶中的第二个活性位点一起使用，以完善密码。由于作为重要蛋白质的tRNA合成酶是新型抗感染药物的靶点，而且哺乳动物细胞中特定的合成酶片段参与信号转导途径，因此可以用于治疗应用，因此拟议的研究加强了健康应用的知识基础。

DESCRIPTION (provided by applicant): The genetic code was established over two billion years ago. Transfer RNAs and aminoacyl tRNA synthetases are at the heart of the code. The synthetases catalyze the attachment of each of the twenty amino acids to their cognate tRNA. Because each tRNA harbors the anticodon triplet for a particular amino acid, the aminoacylation reaction establishes the connection between a nucleotide triplet and a particular amino acid. Transfer RNAs are composed of two major domains that are conserved through evolution. The domains are arranged in three dimensions in an L-shaped structure. One domain is the minihelix, a 12 bp hairpin helix that at the 3'-end is single-stranded and contains the amino acid attachment site. The second domain is positioned at right angles to the first and is a stem-biloop, with one of the loops encoding the anticodon triplet. These two domains are thought by many to have arisen independently. Experiments are designed to explore, in effect, how to make the transition from a minihelix to the genetic code, using noncovalent interactions between the two domains as a way to connect the triplets of the code to an attached amino acid. Because aminoacylated RNA minihelices are considered possible progenitors of aminoacyl tRNAs, a second goal is to establish systems for peptide synthesis based on minihelices. These systems are meant to illustrate at least a few ways to make the transition from the putative RNA world to the theatre of proteins. They also recreate some features of contemporary protein synthesis, including the possibility of reactions dependent on an RNA template. A third objective is to understand the RNA-dependent amino acid discrimination that makes possible the modern universal code. Specifically, experiments are designed to elucidate how subtle information in tRNAs is used with a second active site in synthetases to perfect the code. Because tRNA synthetases as essential proteins are targets for new anti-infectives, and because fragments of specific synthetases in mammalian cells are involved in signal transduction pathways and therefore can be exploited for therapeutic applications, the proposed research strengthens the base of knowledge upon which health applications can be made.