Construction and use of E. coli Nonsense Suppressor Bank

大肠杆菌无义抑制库的构建及使用

• 批准号: 8716260
• 负责人: John Abelson
• 金额: $ 20.86万
• 依托单位: California Institute of Technology
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-06-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8716260&HistoricalAwards=false
• 关键词: Construction; use; E.; coli; Nonsense

This proposal is aimed at constructing synthetic tRNA nonsense suppressor genes, which will direct the in vivo synthesis of a series of nonsense suppressors. Each suppressor will insert a different amino acid in response to a nonsense (in most cases UAG) codon. 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 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 replacement s 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.

该提案旨在构建合成的tRNA无义抑制基因，指导一系列无义抑制基因的体内合成。 每个抑制器都会插入不同的氨基酸来响应无义（在大多数情况下是 UAG）密码子。 目标是衍生出一组能够将 20 个氨基酸中的每一个插入到 UAG 位点的抑制子。 这将极大地有助于蛋白质中氨基酸取代的系统研究，因为 UAG 突变可以通过体内和体外方法在蛋白质的任何位置引入。 从几乎无限的氨基酸取代中获得的信息将有助于蛋白质工程实验。 合成基因由一系列寡核苷酸组合在一起，并通过质粒载体导入大肠杆菌细胞。 通过定点诱变、随机诱变和选择以及构建不同 tRNA 分子的杂交体来引入额外的修饰。 每个抑制子插入的氨基酸是通过在编码二氢叶酸还原酶的基因中采用琥珀突变的系统中的蛋白质测序来确定的。 将寻找通过限制非同源合成酶例如谷氨酰胺tRNA合成酶的误充电来提高某些抑制子的特异性的突变体。 将使用不同的载体系统携带抑制基因，以增加抑制基因对不同研究者的通用性。 该提案的目标是促进蛋白质中特定氨基酸替换的工程化。 原则上，新的氨基酸可以通过称为定点诱变的化学程序插入到克隆基因产物的任何位点。 然而，当需要大量特定替换时，此过程变得很费力。 Miller 博士与加州理工学院的 John Abelson 博士合作，打算通过构建将执行抑制子的转移 RNA 基因来规避这个问题，该基因将被构建为允许在无义密码子 UAG 出现时插入 20 种不同氨基酸中的任何一种。 由于该密码子可以在克隆基因的任何位置创建，因此几乎所有氨基酸替换都是可能的。 这组抑制基因对于科学家研究蛋白质结构和功能之间的关系非常有用。 还将获得有关转移 RNA 分子功能的重要信息。