boxA及上游序列缺失突变对λ噬菌体抗转录终止功能的影响

The effect of boxA and upstream sequence mutation on lambda phage transcription antitermination

目的 研究λ噬菌体表达调控机理 ,阐明PL操纵子boxA及邻近序列突变对抗转录终止作用的影响。方法 利用体内同源重组技术 ,使大肠杆菌染色体DNA上携带了CI85 7,PL ,nutL[box A ( 代表不同的boxA及临近序列缺失突变 )、boxB],N lacZ ,ttt,galK单拷贝基因 ,构建了含boxA及邻近序列突变 (boxA )的一系列新菌株 ,模拟了λ噬菌体感染宿主菌后的生理状态 ,利用抗转录终止报道基因galK分析研究了boxA及临近序列突变对PL操纵子抗转录终止作用的影响。结果 证明在缺失了boxA及部分上游序列的情况下 ,大肠杆菌RNA聚合酶不能通读转录终止子。结论 λ噬菌体左向操纵子具有与右向操纵子不同的转录调控现象。

Objective To study the mechanisms of transcriptional regulation and the effect of boxA as well as boxA upstream sequence mutation on lambda phage transcription antitermination. Methods The recombinant plasmids carrying a set of boxA and upstream sequence deletion were constructed by PCR and recombination DNA technique. To eliminate the artificial effect in the experimental system and mimic the natural state of the bacteriophage lambda, we introduced part of the plasmid sequence [CI857 PL N nutL (boxA boxB) lacZ ttt galK] into E.coli chromosomal DNA with an in vivo recombination method. The effect of boxA and its upstream sequence mutation on lambda phage N mediated transcriptional antitermination were analyzed by galK assay in presence or absence of N under the 42℃ condition. Result All the mutations were defective for antitermination whatever the short deletions removing just boxA or longer deletion that remove upstream sequences in addition to boxA. Conclusion These results suggested that the transcription regulation mechanisms may be different between PL and PR operator. [