Collaborative Research: Construction of E. coli Nonsense Suppressor Bank

该建议旨在构建合成的tRNA无义序列 抑制基因，这将指导在体内合成的一个 一系列的废话（在大多数情况下UAG）condon。 目标是 导出一组抑制器，能够插入20个 在UAG位点的氨基酸。 这将大大有助于系统化 蛋白质中氨基酸取代的研究，因为UAG 突变可以通过体内和体外方法引入， 蛋白质中的任何位置。 从虚拟世界中获得的信息 无限制的氨基酸替换将促进蛋白质 工程实验 合成基因被放在一起 从一系列寡核苷酸中分离并导入E.杆菌 通过质粒载体转染细胞。 补充修改内容 通过定点诱变、随机诱变 然后选择，并通过构建不同的杂交种， tRNA分子。 每个抑制子插入的氨基酸是 通过蛋白质测序在采用 编码二氢叶酸还原酶的基因中的琥珀突变。 将寻找改进某些突变体的特异性的突变体。 抑制器通过限制非同源的误充电 合成酶，如谷氨酰胺tRNA合成酶。 不同的向量 系统将用于携带抑制基因， 抑制器对不同的 investigators. 本提案的目标是促进以下工程： 蛋白质中的特定氨基酸替换。 原则上，新 氨基酸可以插入到重组蛋白产物中的任何位点， 克隆基因的化学过程称为定点 诱变 然而，当出现以下情况时， 需要大量的特定替换。 在 与加州研究所的John Abelson博士合作 米勒博士打算绕过这个问题， 构建转移RNA基因， 体内置换。 转移RNA基因，称为 抑制器，将被构造成允许插入任何 20种不同的氨基酸之一， 发生UAG。由于这个密码子可以在任何位置产生， 克隆的基因，几乎所有的氨基酸替换将是 可能 这种抑制基因的集合将是非常 对科学家研究 蛋白质的结构和功能。 的重要信息 还将获得转移RNA分子的功能。

This proposal is aimed at constructing synthetic tRNA nonsense suppressor genes, which will direct the in vivo synthesis of a series of nonsense (in most cases UAG) condon. The goal is to derive a set of suppressors capable of inserting each of the 20 amino acids at a UAG site. This would greatly aid systematic studies of amino acid substitutions in proteins, since UAG mutations can be introduced by in vivo and in vitro methods at any position in a protein. The information gained from virtual unlimited amino acid substitutions would facilitate protein engineering experiments. The synthetic genes are put together from a series of oligonucleotides and introduced into E. coli cells by a plasmid vector. Additional modifications are introduced by site-directed mutagenesis, by random mutagenesis followed by selection, and by constructing hybrids of different tRNA molecules. The amino acid inserted by each suppressor is determined by protein sequencing in a system that employs an amber mutation in the gene encoding dihydrofolate reductase. Mutants will be sought which improve the specificity of certain suppressors by limiting the mischarging by non-cognate synthetases, such as glutamine tRNA synthetase. Different vector systems will be used to carry the suppressor genes to increase the general utility of the suppressors to different investigators. The goal of this proposal is to facilitate the engineering of specific amino acid replacements in proteins. In principle, new amino acids can be inserted at any site in the product of a cloned gene by a chemical procedure known as site-directed mutagenesis. However, this procedure becomes laborious when large numbers of specific replacements are needed. In collaboration with Dr. John Abelson at the California Institute of Technology, Dr. Miller intends to circumvent this problem by constructing transfer RNA genes that will perform the substitutions in vivo. The transfer RNA genes, known as suppressors, will be constructed so as to allow insertion of any one of the 20 different amino acids whenever the nonsense codon UAG occurs. Since this codon can be created at any location in a cloned gene, virtually all amino acid replacements will be possible. This collection of suppressor genes will be extremely useful to scientists studying the relationships between the structure and function of proteins. Important information on the functioning of transfer RNA molecules will also be obtained.