Thioredoxin活性中心氨基酸取代對噬菌體T7及線形噬菌體生長的影響

Abstract

摘要 Thioredoxin在不同 類型的細胞中扮演不一樣的角色. 其最早被以核酸還原酵素的輔因子發 現, 近來則發現其對大腸桿菌線形噬菌體f1, fd, M13等的組合及T7噬菌 體的生長 為必須的. T7 DNA 聚合酵素由噬菌體的基因5蛋白質及 Thioredoxin所構成. 在線形噬菌體中Thioredoxin可能與基因I蛋白質進 行交互作用推動噬菌體組合作用. 然而, 在不同型的蛋白質-蛋白質交互 作用中, 對Thioredoxin所扮演的角色依然所知不多. 為了了解大腸桿 菌Thioredoxin基因中不同氨基酸取代對T7及線形噬菌體生長的影響, 我 們利用PCR-定點突變法將Gly33以Asp取代建構一位於Thioredoxin活性中 心的突變株, 藉由生體內分析-噬菌體對突變株感染的能力來分析突變株 的特性. 結果顯示, Thioredoxin Gly33的位置引入帶電荷氨基酸的突變 對線形噬菌體生長有很大的影響, 對T7噬菌體生長的影響則較小. abstract Thioredoxin appears to play a variety of roles in different cell types. It was isolated initially as a co-factor for the reduction of ribonucleotide diphosphatesby ribonucleotide reductase, has recently been shown to be required for the assemblyof the filamentous Escherichia coli phages f1, fd, M13, and required for phage T7growth. T7 DNA polymerase is composed of the gene 5 protein of the phage and Thioredoxin. Interaction has been predicted as an engine for phage assembly between the geneI protein of filamentous phage and thioredoxin system. However, little is known of of the role of thioredoxin in different" protein-protein interaction" types. In order to understand the influence of the different amino acid substitutionsin E.coli thioredoxin on the growth of bacteriophages T7 and filamentous phages , we have constructed a mutant in the thioredoxin gene changing its catalytic core.PCR site-directed-mutagenesis was crrried out to replace conservative Gly33 by Asp. The mutant was characterized using an in vivo assay based on the ability of cellto support growth of T7 and filamentous phages. The results indicated that the charged group side-chain in the position 33 of amino acid residues have great effecton the growth of filamentous phages, and little effect on the growth of T7 phages.