大腸桿菌Thioredoxin突變株與f1線形噬菌體的 Gene I蛋白之交互作用

Abstract

Thioredoxin存在許多不同型態的細胞當中，且具有許多功能的熱穩定蛋白，在不同的細胞型態中扮演不同的角色。在大腸桿菌內當作是一般的蛋白質雙硫鍵的還原劑。Thioredoxin亦為噬菌體T7和線形噬菌體f1在感染大腸桿菌時所必須。 缺乏thioredoxin時，f1 可以正常的表現蛋白質，也可正常的複製其DNA，但是無法組裝其子代。一些遺傳證據顯示，thioredoxin可能會與f1的Gene I蛋白作用。 本論文一方面利用LexA酵母菌雙雜交系統來進一步證實thioredoxin與Gene I間的交互作用，但可能因為融合的蛋白沒有正常的摺疊、融合後遮蔽了交互作用的區域或者此兩蛋白的交互作用不適合於LexA酵母菌雙雜交系統，所測得的結果顯示無交互作用。另一方面我們篩選可恢復在 G33A、G33T、G74N、G74S、G74T和G92A生長的Revertant噬菌體，定序後發現Revertants的突變點皆位於Gene I的第142個胺基酸，除G74T的Revertant由Asn變為His外，其餘皆變為Tyr，與gfip的突變相同。 就基因上的觀點，我們認為Thioredoxin與Gene I蛋白有交互作用，但可能無法用LexA酵母菌雙雜交系統測得。就結構而言，Thioredoxin的第31、32、33、34、74和92位置Gene I的第142個位置應不會對應到同一個Gene I的位置，推測Gene I的Asn 142可能不是直接作用的位置，但對Gene I上的thioredoxin結合區域之結構是很重要的。

Thioredoxin is a multifunctional heat stable protein found in all cell types. It appears to play a variety of roles in different cell types. In E. coli it is a general protein disulfide reductant, can serve as the reductant for synthesis of deoxyribonucleotides, is involved in sulfate metabolism, and is required for the reduction of methionine sulfoxide. Thioredoxin is required when phage T7 and filamentous phage f1 infect E. coli. When lacking thioredoxin, protein expression and DNA replication of f1 are normal but can not be assembled into virions. Thioredoxin may interact with f1 Gene I protein in some genetic evidence. In this study, the LexA Yeast Two Hybrid System was used to prove the interaction between thioredoxin and Gene I protein, but result turn out negative. It is likely due to the misfolding of the fusion protein, the fused protein occluding the site of interaction, or unknown reasons. We have isolated mutants that restore the infectivity of f1 in E. coli. thioredoxin G33A, G33T, G74N, G74S, G74T and G92A mutants. The revertant for G74T is Gene I N142H. The others are Gene I N142Y, the same mutation as gfip that was isolated by Ressel M. and Model P. in 1983. We believe that thioredoxin interacts with Gene I protein based on genetic information. But it may not be detected by LexA Yeast Two Hybrid System. According to the structure, the amino acid 31,32, 33, 34, 74 and 92 of thioredoxin should not interact with the same amino acid of Gene I. So Asn-142 seems to be the important site for the structure of thioredoxin binding domain in Gene I, but not directly involved in interaction.