Construction and use of E. coli Nonsense Suppressor Bank

本课题旨在构建合成tRNA无义抑制基因，指导一系列无义抑制基因的体内合成。每个抑制子都会插入一个不同的氨基酸来响应无义密码子（在大多数情况下是UAG）。目标是获得一组能够在UAG位点插入20个氨基酸中的每一个的抑制子。这将极大地帮助系统地研究蛋白质中的氨基酸取代，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替换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，通过质粒载体导入大肠杆菌细胞。其他修饰包括位点定向诱变、随机诱变后选择、以及构建不同tRNA分子的杂交体。在编码二氢叶酸还原酶的基因中采用琥珀色突变的系统中，通过蛋白质测序确定每个抑制子插入的氨基酸。将寻求通过限制非同源合成酶（如谷氨酰胺tRNA合成酶）的误充来提高某些抑制因子特异性的突变体。将使用不同的载体系统来携带抑制基因，以增加抑制基因对不同研究人员的通用效用。这一建议的目标是促进特定氨基酸替代蛋白质的工程。原则上，新的氨基酸可以通过一种称为位点导向突变的化学过程插入到克隆基因产物的任何位点上。然而，当需要大量特定的替换时，这个过程变得费力。在与加州理工学院的John Abelson博士的合作中，Miller博士打算通过构建执行抑制子的转移RNA基因来规避这个问题，该基因将被构建成允许在无义密码子UAG出现时插入20种不同氨基酸中的任何一种。由于这个密码子可以在克隆基因的任何位置产生，因此几乎所有的氨基酸替换都是可能的。这组抑制基因对研究蛋白质结构和功能之间关系的科学家非常有用。还将获得有关转移RNA分子功能的重要信息。

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.