nut R 中的突变对λ噬菌体早期转录抗终止作用的影响

Mutations in nutR Affect Early Transcription Antitermination in Bacteriophage λ

将λDNA的nutR序列置于Lac启动子的下游,在nut R和β-半乳糖苷酶基因(gal K)之间插入λ噬菌体依赖rho的终止子tR1,使ga1 K的表达取决于N蛋白介导的转录抗终止作用。为研究nutR对抗终止的影响,系统地进行了该序列中每个碱基的点突变(A→C,G→T,C→A及T→G)。结果表明boxA中有两个碱基(位置2和5)的突变对抗终止作用是至关重要的,使抗终止效率降低了10倍;而其他位置的改变影响甚微。boxA的缺失在nus^+宿主中使抗终止效率降低了40％,但在nusB宿主中却恢复到野生型水平。boxB茎环结构中,茎部顶端的碱基对及环中邻近基部的两个碱基的突变对抗终止作用影响很大,被认为是N蛋白的识别序列。boxA和boxB之间的间隔序列中有两个碱基的突变几乎使抗终止作用丧失。

In this study, to examine the role of transcription antitermination and nut site function, we have placed the nutR region downstream of a lac promoter. Between nutR and galK, the X rho-dependent terminator, tR1 , has been placed, so that the expression of galK depends on transcription antitermination by N protein. We havt- sys tematically changed each base (A→C, G→T. C→A, or T→G) in nutR site to examine its effect on antitermina tion. The results indicate that only positions 2 and 5 of the 9 base pairs of boxA were found to be critical. The point mutation of these two positions decreases N antitermination about 10- fold. Other positional changes remained functional or caused minor decrease. Deletion of boxA decreases antitermination about 40% in nus+ host strains. However, NusB does not exert an effect on N-dependent transcription antitermination when the boxA seg-ment is deleted. We have also found that every base changed at any position within the 5 base boxB loop is unfavorable to antitermination by the N protein of bacteriophage X. When the T-A base pair at the top of boxB stem and the stem-adjacent two bases of the loop are changed, it causes a serious prevented in antitermination. The segment may be sequence specific for N recognition. Mutation of two positons of the 8 base interval between boxA and boxB causes a defect in antitermination.